NPC Valorisation Voucher - Netherlands Proteomics Centre

2346824262728293031323536FrontpageSkin secretions of amphibians provide a rich source of bioactiveContentspeptides (see article pg 8).AboutPrefaceNews headlinesInterview: Bert PoolmanHighLights8 Martijn Pinkse, Geisa Evaristo, Mervin Pieterse and Peter Verhaert; How to discover bioactive peptides easierand faster12 Arjen Scholten, Christian Preisinger and Albert Heck; Investigating perturbations induced by kinaseinhibitor Gleevec16 Serena Di Palma and Shabaz Mohammed; Accelerating towards ultrasensitive proteome analysis20 Teck Yew Low, Flore Kruiswijk, Roberto Magliozzi and Daniele Guardavaccaro; Genotoxic stress affectsprotein synthesis through regulation of eEF2KBlighted by Kenning: Apples infected with knowledgeNPC Valorisation Voucher in practiceNovel biomarkers patent filedValorisation for scientistsBioBusiness Summer SchoolDTL updateNPC Supervisory BoardNPC Top Publications10 Years NPC: Progress in Proteome BiologyEduard Klasen: Golden future16 | NPC Highlights 16 | November 2012>AboutThe Netherlands Proteomics Centre (NPC) is a strategic collaboration of research groups fromseven universities, four academic medical centres and several research institutes and biotech companies.With a scientific programme addressing key areas of proteomics in several projects, andspecialised ‘research hotels’, the NPC performs high-quality research and knowledge transfer in aninternational context. The NPC is part of the Netherlands Genomics Initiative.In NPC Highlights researchers present progress and results from NPC projects of the scientificprogramme and the research hotels. NPC Highlights is published by the Netherlands ProteomicsCentre.Netherlands Proteomics CentrePadualaan 8NL 3584 CH Utrechtt +31 30 253 4564e info@npc.genomics.nlw www.netherlandsproteomicscentre.nlEditorial BoardAlbert Heck, Scientific Director NPCWerner Most, Managing Director NPCBert Poolman, member Executive Board NPCHerman Overkleeft, member Executive Board NPCRob Liskamp, member Executive Board NPCGeert Kops, member Executive Board NPCMartje Ebberink, Communications NPCText interviewsAstrid van de Graaf, Esther Thole, Lilian Vermeer,Bastienne WentzelCoordination and editingMarian van OpstalBèta Communicaties, The HagueLay-outFrans KoemanF.Koeman DTP-Services, ZoetermeerPhotographyBas van Breukelen, Thijs Rooimans, Henk VeenstraPrintingBestenzet BV, ZoetermeerTo subscribe to NPC Highlights pleasesend an e-mail with your full name, organisationand address to info@npc.genomics.nl© 2012 Copyright Netherlands Proteomics Centre

Welcome It is my pleasure to presentsome of the current NPC highlightsaddressing scientific research, valorisation,education and science communication.This issue nicely demonstrates the broadspectrum of activities endorsed by theNPC.To start, UK-based artist Charlotte Jarvis has elaborated onthe bio-art project ‘Blighted by Kenning’, together with NPCresearchers and our communication team, over the last year.This summer we unveiled ‘Blighted by Kenning’ in Suffolk, UK. Itwas a great success and we are proud to announce that you cannow visit the exhibition, which runs until 6 December at HôtelDroog in Amsterdam. The NPC also participates in the D&A popupstore, a new concept for public communication coordinatedby the Netherlands Genomics Initiative. A small installation of‘Blighted by Kenning’ will be exhibited at the D&A store as of15 November 2012. More information about this exciting projectcan be found on page 24.Another example of our activities is the strong focus on thetraining and education of young, talented researchers withinthe NPC. As in previous years, this summer a couple of PhDstudents received an NPC grant to visit the EU Summer Schoolin Proteomic Basics in Italy. Furthermore, five NPC researchersvisited the BioBusiness Summer School in Amsterdam. You canread about their experiences on page 29.We are also looking forward to the coming years. On 11&12February 2013 we will be celebrating the 10 th anniversary of theNPC where a great number of internationally renowned speakerswill give lectures in addition to other activities. Please visit theNPC website for details of the programme as well as for abstractsubmission and registration, and mark your calendar to make sureyou will be there!| The NPC is now busy preparing a strategy for the future. Partof this is a more integrated approach to biological research bymultiple enabling technologies as proposed in the concept of theDutch Techcentre for Life Sciences (DTL) of which NPC is one ofthe founding partners. You can find an update of the initiation ofDTL on page 30.We hope you enjoy reading this issue, and please feel free tocontact us any time for any questions or suggestions.Albert Heck, scientific director NPCpreface

News HeadlinesNPC Valorisation VoucherCall for applicationsResearchers in proteomics with early stage commercialisationplans are invited to write a proposal for an NPC ValorisationVoucher. The NPC Valorisation Voucher is meant to enableproof-of-concept studies and fill in the gap towards furtherfunding of commercialisation plans (e.g. NGI Pre-Seed).Projects qualifying for a Valorisation Voucher could addressexperimental validation, marketing and/or IP related support,required to build a solid business opportunity.For more information please go to our website or contactAdinda Woelderink, NPC Valorisation Managerwoelderink@npc.genomics.nl/+31 30 253 6803. | NPC Highlights 16 | November 2012Nature publicationDeletion of genes causes cancerIn a recent Nature paperHans Clevers’ group of theHubrecht Institute togetherwith researchers from the UMCUtrecht, Utrecht University andthe Netherlands ProteomicsCentre describe a gene thatlimits the growth of intestinaladenomas. The research mightyield a lead compound inthe treatment of intestinal cancer. Stem cells in the gutcontinuously provide new tissue. Hans Clevers succeededearlier in identifying and isolating these stem cells. Togetherwith Madelon Maurice (UMC Utrecht) and Albert Heck (UU,NPC), the Clevers group searched for genes which are onlyactive in the intestinal stem cells. They found RNF43. Whenthis gene is deleted, exponential growth of the intestinal stemcells cause adenomas, a pre stage of intestinal cancer.Publication: Tumour suppressor RNF43 is a stem-cell E3ligase that induces endocytosis of Wnt receptors (Nature,30 augustus 2012, 488, 665–669; doi:10.1038/nature11308)Visit DTL onlineThe Dutch Techcentre for Life Sciences (DTL) is a collaborativepublic-private initiative to provide high-end and pioneertechnology to enable ground-breaking research in molecularbiosciences.In essence, DTL is a strongly intertwined network of expertcentres among the Dutch universities, university medicalhospitals as well as public and private research institutes. NPCis one of these expert centres and founding partner of DTL. Tosupport the development and branding of DTL, the Communityand Outreach Team developed a logo and website for DTL(www.dtls.nl).In addition, a LinkedIn groupis opened for discussions.Please join!Shopping for DNAThe Netherlands GenomicsInitiative has launched a newconcept: the D&A pop-up store.The store pops up in an emptyvenue in an arbitrary Dutch city’sshopping street, stays there for ashort time, and then disappearsagain to pop up in another citysome days or weeks later. TheD&A store travels through the Netherlands until April 2013 andtries to acquaintance as many people as possible with DNA.Visitors can experience DNA, not just as a genetic profile, butas a lifestyle. The NPC participates in this project with a smallinstallation of the bio-art project Blighted by Kenning.Visit the website of the D&A store to see if the store also popsup in your city!: www.dnastore.nlNPC PhD Day 2013The next edition of the NPC PhD day is coming up on 17January 2013. The NPC is organising a day of lectures, workshopsand a lab tour in collaboration with DSM BiotechnologyCenter in Delft. All NPC affiliated PhD students are invited toattend this day. Attendance is free of charge, but those interestedshould RSVP by December 3 rd . For more information, theprogramme and registration, please visit the NPC website.

awards honours newsSecond DA4GA exhibitionThe Designers & Artists 4 Genomics Award(DA4GA) highlights and explores the excitingand novel possibilities between design,artistic practice and life sciences. Conceivedby the Netherlands Genomics Initiative, CSGCentre for Society and the Life Sciences,Naturalis, and Waag Society, DA4GA aims tostimulate emerging designers and artists todelve into the world of bio-art, and producenew work in close collaboration with the Netherlands mostprestigious Life Sciences research institutes, for example in thefields of sustainability, food, health, bio-informatics, agriculture,safety and energy. The second edition of Designers & Artists 4Genomics is currently showcased in museum Naturalis in Leiden.They are on exhibition until the end of this year.Dolf Weijerspersonal professorDolf Weijers has been appointed fullprofessor with a personal Chair inBiochemistry of Plant Development.The appointment to personal professoris made on the basis of greatmerit in scientific research andeducation, as well as remarkablepersonal qualities. Weijers is abiochemist and developmental biologistand he is involved in NPC’s Research Theme ‘ProteomeBiology of Plants’ (T2). Weijers (1976) studied biochemistry atthe Hogeschool Enschede and in 2002 received his PhD on thehormonal control of embryo development in plants from theUniversity of Leiden. His studies focus on initial constructionof stem cells in plant embryos. For his work on the constructionof tissues and stem cells in plants-embryos he received aVIDI grant from NWO (2006) and an ERC Starting Grant (2011).He is also a member of the Young Academy - KNAW.Dialogue betweenscience & societyThe CSG Centre for Society and the LifeSciences invites all interested to ShakingScience!: the event to meet the lifesciences. All on the principal that sciencemeets society and society meets science.During the full month of November 2012visitors can attend workshops, films,debates, lectures and expositions throughout the country wherenew technology and society meet each other. Activities thatinspire and provide food for thought. The NPC participates inthis event with the bio-art project Blighted by Kenning.Learn more about Shaking Science!: www.shakingscience.nlNovel mass analyser unravelscomplex biomolecular structuresEssential for researchand development oftherapeuticsIn a recent Nature Methods publication NPC scientific directorAlbert Heck and his colleagues from Utrecht Universityintroduced a new mass spectrometer based on Orbitrap TMtechnology. This high-sensitive instrument might play a crucialrole in the development and use of therapeutic antibodies.In close collaboration with the research group of AlexanderMakarov of Thermo Fisher, the inventor of the Orbitrapanalyser, the reseachers show that protein assemblies ofmolecular weights over 1 million Da can be analysed with veryhigh analytical resolving power and exquisite sensitivity downto detection of single ions. The new mass spectrometer allowsthe measurement of a range of important proteins and proteinassemblies allowing a detailed analytical footprint of thesebiologically and medically important molecules. Especiallyin the fast-growing arena of biopharmaceuticals (e.g.therapeutic antibodies) this new instrument will be importantboth in research & development and in quality control, toenable such molecularly complex biomolecules to be usedsafely in the clinic.Heck: “This new mass spectrometer opens up avenues tomeasure not only protein-protein interactions, but alsocovalent and non-covalent binding of small molecules toprotein assemblies. Wide-ranging applications may includethe direct analysis of drug molecules binding to their targets,and the investigation of post-translational and chemicalmodifications on intact proteins and protein assemblies.”NPC Valorisation WebsiteLooking for more informationon ways to utilise your knowledge?Plans for starting yourown company? Looking for(business) events and courses?In search of funding optionsfor early startups?Check out our website! Overthe last months the NPCoffice has been working on the website focused on valorisation.The objective of the website is to give more insight intothe NPC valorisation activities and to provide the NPC researcherswith information on how to ‘valorise’ their research.The NPC valorisation website is part of the NPC website.www.netherlandsproteomicscentre.nl/npc/valorisation.html|

Interview|Bert Poolman“Building a complete cell is theultimate goal many researchers areafter,” says Bert Poolman, professorof Biochemistry at the University ofGroningen. On the path to this stilldistant goal, he and his researchgroup try to unravel and understanddiverse membrane translocationprocesses, notably transport ofmolecules in and out of cellsand their organelles. One majorcurrent objective is to gain furtherunderstanding of the biogenesisof membrane proteins and theunderlying trafficking mechanisms. | NPC Highlights 16 | November 2012Proteomics greatly helpsin finding the right research pathMembrane proteins are involved in many essential cellularprocesses such as transport of nutrients, sensingof environmental changes, energy transduction andscaffolding of cell structure. Due to their important roles inthese processes, they are also linked to various diseases thatmake them clinically important as potential drug targets. BertPoolman’s research group Membrane Enzymology (part of theGroningen Biomolecular Sciences and Biotechnology Institute(GBB) and the Zernike Institute for Advanced Materials)has accumulated a great deal of experience in the field ofmembrane translocation processes and membrane proteins ingeneral.Poolman explains: “Our research may generate applicationsfor the drug industry, for example, but our focus is mainlyfundamental; we investigate which molecules are involved intransport and try to unravel how they function. Proteomics hashelped to determine the focus of our research. For example,if via DNA microarrays we find that the expression of hundredsor thousands of genes in a cell have changed in response toa particular stress, then using proteomics we can find outwhat this means at the level of proteins and narrow down thenumber of relevant targets. If you are able to reproduce thesechanges by engineering of the cell, then you really begin tounderstand the mechanisms behind the changes.”Poolman’s team managed to successfully engineer the testorganisms L. lactis, E. coli and S. cerevisiae, which haveproven to be suitable for hosting the production of membraneproteins.NPC Research HotelMany processes in membrane transport still need to be unravelled.“We are not able to change the characteristics of membraneproteins rationally, let alone to adjust the transportmechanisms in a cell,” states Poolman. In his research careerhe hopes to find that more directed interventions in the functioningof a cell — the field of synthetic biology — are possible.The work complements Poolman’s fascination with unexpecteddiscoveries. “I always tell my students and postdocs to stayalert for deviations in measurements and not to stick to prevailingdogmas. This may lead to unexpected breakthroughs.”

“I always tell my students not to stickto prevailing dogmas. This may lead tounexpected breakthroughs.”CareerMany research groups have collaborated or are still collaboratingwith the Poolman group because of its expertise in membranebiology. Being an NPC Research Hotel generated a greatdeal of collaboration as well. NPC guest researchers can haveproteins from highly complex mixtures analysed by mass-spectrometry-basedidentification, or individual membrane proteins,protein complexes and their modifications can also becharacterised biophysically using varying techniques. “Sincemembrane transport occurs in diverse cellular processes, wehave dealt with various topics outside of our own researchfocus as well.”Super-resolution optical microscopyTo study the dynamics and localisation of processes in cells,Poolman’s group uses super-resolution microscopy and otheroptical methods, developed with Professor Antoine van Oijen’sMolecular Biophysics group at Groningen. “Using standard opticalmicroscopy techniques, particles smaller than 200-300 nmcannot be discerned. However, with super-resolution one candiscern particles down to 10-20 nm, which is in the size rangeof proteins,” explains Poolman. “Thanks to this technique wewere able to discover that membrane proteins (coloured witha fluorescence label) are not homogeneously divided over themembrane but reside in clusters. The clustering of proteinsmay cause different properties. So far, we have been studyingensembles of cells, but our goal is to determine the proteomeof a single cell. Using this process one can really determinethe differences between individual cells.”Recent measurements indicate that seemingly homogeneousbacterial populations are in fact heterogeneous. “We wantto know why they are heterogeneous and what causes thisheterogeneity. Stochastic processes may play a role; sometimescertain proteins just happen to be present in a lowerconcentration in one cell than in the other. But occasionallythere’s a more sophisticated reason behind it. For example,if some cells are more resistant to high saline conditions thanothers, their osmoregulatory transporters may be different.If post-translational modifications are involved or moleculesare embedded in different lipid environments, two proteinsidentical in amino acid sequence may each have a differentactivity. High-resolution optical microscopy on the one handand high-resolution mass spectrometry on the other will bekey to elucidating such differences.”Continuation requiredTo perform all this research and help other researchers withtheir membrane protein questions, the Poolman group usesexpensive equipment worth more than 1 million euros. “Tostay up-to-date in our field, we occasionally need to purchasenew equipment, which obviously also costs a lot. Furthermore,to hold on to good proteomics experts we need to offer themBert Poolman2008 Program Director Centre for Synthetic Biology2003 Visiting professor at the California Instituteof Technology, Pasadena, U.S.A.1999 Professor of biochemistry, University ofGroningen1990 Research fellow at the Royal NetherlandsAcademy of Arts and Sciences (KNAW)1987 PhD degree (cum laude), University ofGroningenAwards2011 Elected as Faculty 1000 member2010 NWO Top-GO subsidy on membrane proteinbiogenesis2009 Elected as member of the Royal NetherlandsAcademy of Arts and Sciences (KNAW)2008 NGI grant renewal (NPCII) for the MembraneProteomics Centre2007 NWO Top subsidy to support the research onABC transporters and cell volume regulationRecent key publications• Erkens, G.B. et al. (2011) The structural basis ofmodularity in ECF-type ABC transporters. NatureStruct. Mol. Biol., 18, 755-760.• Karasawa, A. et al. (2011) Cystathionine b-synthetase(CBS) domains 1 and 2 fulfill differentroles in ionic strength sensing of the ATP-bindingcassette (ABC) transporter OpuA. J. Biol. Chem.,286, 37280-37291.• Mika, J.T., and Poolman, B. (2011)Macromolecule diffusion and confinement in prokaryoticcells. Curr. Opin. Biotech., 22, 117-126.• Marreddy, R.K., et al. (2010) Amino acid accumulationlimits the overexpression of proteins inLactococcus lactis. PloS One, 5, e10317.steady jobs. Otherwise they very quickly start looking fornew jobs. The NPC has helped us enormously in providingthe means for this and structuring proteomics research inthe Netherlands. However, after 2013 when funding by theMinistry of Economics for NGI and consequently for NPC isdiscontinued, financial support will be needed to continueresearch at a high level.” Poolman has good hopes that a newinitiative like the Dutch Techcentre for Life Sciences (DTL)will be able to provide these means and secure the frontlineposition of Dutch R&D. Poolman hopes that the name NPC cansomehow be maintained, “This has really become a wellknownbrand name.”|

Martijn Pinkse, Geisa Evaristo, Mervin Pieterse and Peter VerhaertHow to discover bioactivepeptides easier and faster | NPC Highlights 16 | November 2012Unnecessary efforts in structural analysis and bioassayscreening are no longer a prerequisite for recognising themost promising peptides from a complex natural sample.Two-dimensional display of liquid chromatography andtandem mass spectrometry runs in combination withspecific post-translational chemical modifications caneasily identify peptides with potential bioactivity. Themethod is demonstrated on the basis of the skin secretionsof amphibians, which has already led to the discovery ofseveral novel bioactive peptides.In all of biology, peptides are used as fundamental communicationmolecules in all branches of the tree of life: frommicroorganisms all the way to man. Yet it is only in the lastfew years that the general awareness has steadily been growingof the enormous potential of biologically active peptides,not only as biomarkers for diagnostics but also as (leads for)novel therapeutics. This increased interest is noticeable intoday’s applied sciences, with a clear movement within discoveryresearch of both pharmaceutical and (other) industrialbiotechnological sciences to explore our planet’s biodiversityfor potentially interesting bioactive peptides.The conventional strategy for novel peptide discovery hasbeen the systematic bioactivity screening of complex naturalsources. Bioassays reflecting the activity of interest wereperformed on chromatographically separated fascinating naturalpeptide mixtures. Bioactive fractions were consequentlypurified to homogeneity for primary structure analysis of therespective compounds. Hundreds of biologically active peptideswere discovered this ‘classic’ way and, in many cases,multiple peptides with different bioactivities were discoveredwithin the same natural source [1].Over the past years, the Delft NPC Analytical Hotel has builta unique expertise in peptidomics (the subset of proteomicsfocusing on the low molecular weight proteins), which in ouropinion can play a welcome role in complementing the bioassaybased discovery research for novel bioactive peptides.Focus on bioactivity With its ever increasing sensitivity andspecificity, mass spectrometry (MS) became an invaluable analyticaltool in peptide biology at the very moment it was introducedinto the field. Thanks to MS analyses, it became obviousthat the ‘classic’ discovery strategy is seldom all-inclusive,and that many (if not the majority of) peptides from oftenvery rich biological sources remain unstudied. The number ofbioassays a certain lab can run is after all limited, so manypotentially interesting peptide species are bound to escapethe researchers’ attention, which is particularly a pity in cases

What this research is about:Screening natural sources forpharmaceutical peptidesShort peptides play a crucial role in all sorts of biological processes. They take care of the communication inthe body, but can also have an antibiotic, antiviral or fungicidal activity. Therefore peptides have high pharmaceuticalpotential. “Many peptides have not yet been discovered. They are very often missed by conventionalproteomics methods that are not designed to detect them,” says Peter Verhaert, professor of AnalyticalBiotechnology and Innovative Peptide Biology at Delft University of Technology. “Besides that, the classic studyof peptides in natural samples is still a very laborious and time-consuming task. If we were able to recognisepeptides that have promising pharmaceutical potential at an earlier stage, it would save a great deal of effort.”In this article Verhaert and co-workers describe a novel generic method for discovering peptides with bioactiveproperties in complex mixtures obtained from natural sources which would otherwise remain unrevealed.He uses a combination of liquid chromatography with tandem mass spectrometry analysis and when necessarychemical modifications to identify potentially bioactive peptides. “We look at a set of typical structuralelements that are indicators for bioactive compounds. Sometimes we make those visible by giving the samplea chemical treatment and by comparing it with the untreated sample,” explains Verhaert. After identificationand characterisation of the promising peptides, final proof of the bioactivity has to be demonstrated using abioassay.The method is applicable on all kinds of complex mixtures. Verhaert uses as model system his favouritenatural source: the frog. These animals secrete a wide variety of bioactive peptides via glands in their skin aschemical defence. “Their skin is their only method of protection against all sorts of predators and microorganisminfections. It is an abundant source of undiscovered peptides with bioactive properties, which includepeptides with antimicrobial activity, peptides that inhibit enzymes or paralyse muscles. It also provides peptidesfor accelerating wound repair, which could in time deliver a more effective drug for wound healing forhumans,” Verhaert says.| NPC3 T3: Proteome Biology of Micro-organismswhere the natural supply of peptides is very precious.It is evident that complex biological peptide collectionsconsist of a mix of the actual bioactive ones as well as theirbreakdown products and irrelevant protein-derived ‘contaminants’.The brute force structural elucidation of all the(non-tryptic) peptides is something which may become possiblein the future, but which today is still unrealistic. Havinga system which helps to focus on those peptides that are themost likely to exhibit bioactivity at one’s disposal is thereforeextremely useful.Figure 1 | Lacking mechanical defence systems, frogs, toads andsalamanders are very vulnerable and rely entirely on the activity of thespecific collection of peptides (and other biochemical compounds) secretedon their body surface to cope with environmental hazards, includingmicrobial infection and animal predation [picture of Brazilian walking leaffrog, Phyllomedusa burmeisteri, during peptide secretion ‘milking’ bypeptidomics research scientist].Structural families Looking a bit deeper into the structuralbiology of bioactive peptides, one sees that, other than exhibitinga particular bioactivity, they tend to share special characteristicsthat differentiate them from their inactive counterparts.Indeed, the many bioactive peptides known today canbe grouped into several structural families, reflecting thatcertain structural motifs tend to be common among peptidegroups, as well as conserved between various taxa of organisms.These common conserved structural domains frequentlycoincide with the biologically active site of the peptide under

with TIC in y-axis) does not give much information on a sampleother than a vague indication of its magnitude and complexity.With the publicly available software MSight (www.expasy.org/MSight), LC-MS data are assembled into a more informativetwo-dimensional image (see Figure 2, for details see [3])which can be readily interpreted with respect to structuralfeatures (i.e. peptide PTMs) of interest to the biologist.Figure 2 | A. Orthogonal plot of LC-MS run of amphibian skin secretionsample. Each spot represents an isotope cluster of a peptide-like ion. Insert:zoom-in area of 2D LC-MS plot revealing amidated peptide ions, as slightlymore hydrophobic (increased RT on reversed phase HPLC) and exactly one Dalighter isotopes.B. Similar display of same sample after chemical treatment (i.c. reduction).investigation. This is the part of the peptide which fits in thebinding pocket of a receptor or within a groove of the catalyticsite of an enzyme modifying its activity.In this respect post-translational modifications (PTMs) arecritically important biochemical alterations of a peptide’s10 | NPC Highlights 16 | Peter Verhaertprimary structure, as these often dramatically affect (part of)their tertiary (3D) structure, making it fit its binder or not.Using high-end MS based peptide analytics (employing Q-TOFand orbitrap analysers), we developed methods with sufficientMS resolution to specifically screen for these distinctivefeatures.Easily spotted Some PTMs are visible in untreated samples byvirtue of the fact that they are not present on one hundredpercent of the molecules, which means that for each posttranslationally-modifiedpeptide, the unmodified version isalso often present in a sample. Depending on the type of PTM,these can be seen at specific mass differences and more orless predictable retention time shifts. A PTM easily spottedthis way is C-terminal amidation. Replacement of the freeacid carboxyl end (-COOH) of a peptide to its amide (-CONH 2 )yields a modified peptide which differs exactly one mass unitfrom its unmodified variant, whereas its retention only slightlyshifts (with the amidated version typically a bit more hydrophobicthan the free acid; see insert in Figure 2).Other PTMs only reveal themselves after specific chemicaltreatment of the sample. In these cases the differential 2Ddisplay comparing treated and untreated samples very elegantlyshows peptides with the targeted PTM (see Figure 3).Such treatment can be a very simple one, such as reduction ofthe sample by dithiothreitol (DTT) or an alternative reducingagent. In this way we have been able to identify and fullystructurally characterise novel peptides with both intra- aswell as intermolecular disulfide bridges in various frog andtoad species [3, 4]. Subsequent bioactivity studies thenshowed that several of these newly discovered peptides haveFrog skin secretions A good example of a rich source ofbioactive peptides of sometimes very limited supply is thedefensive secretion by the dorsal skin glands of exotic amphibians(see Figure 1). Nowadays, this material — not infrequentlyhundreds of different peptides per sample — is typically noninvasivelycollected in the wild. As a third of all amphibianspecies are threatened with extinction, it is guaranteed thatpeptide donors remain unharmed and are released back intotheir habitat immediately after ‘milking’ [2]. Depending onthe biological need or ecological niche of a certain amphibianspecies, bioactivities to be found in its skin secretions are verydiverse, and, not seldom, unexpected. Focusing on a limitednumber of bioactivities, therefore, risks missing many potentiallyinteresting compounds.2D mapping We have successfully utilised a two dimensional(2D) peptide display, being an image generated from the 2Dplot of retention times versus mass-to-charge ratios of LC-MS(peptide) ions, to identify peptides with predefined PTMs/structural features indicative of their potential bioactivity. Aconventional one-dimensional LC-MS display (chromatogramFigure 3 | A. MSight overlay of two subsequent LC-MS runs (Fig. 2A and B) ofuntreated and reduced sample of amphibian skin secretion. This generates atwo-dimensional PTM driven differential display, revealing peptides with andwithout cysteine bridges.B. Zoom-in of selected areas focusing on peptides (from left to right panels)without cysteine bridges (far left), with a single disulfide bridge, with twoand with three S-S bonds (far right). Peptides originally carrying a disulfidebond are easily distinguished from the others (non cystine containing peptidesremain at identical RT and m/z in both untreated and treated sample)by their shift in both retention time (vertical axis) and m/z value (horizontalaxis; i.e. increase of 2 Da per disulfide bridge broken in the reduced sample).

anti-microbial effects [3, 4], whereas others can be classifiedamong protease inhibitors [4].General conclusion A common tool used to represent LC-MSdata as two-dimensional maps can be employed to reveal definedstructural features of the MS detected biochemical. Twodimensional(differential) peptide displays are elegant meansfor mining complex natural sources of potentially bioactivepeptides. They help focus on distinctive structural features ofthe peptides, thereby becoming part of the discovery strategyof peptides with bioactivity potential.As such this post-translational modification driven analyticalmethod embodies a complementary ‘reverse’ approach to thebioactivity based discovery of bioactive peptides. One startsby targeting potentially interesting peptides from a natural(microbial or vertebrate) source prior to elucidating their primarystructure, and finally analysing their biological activity/pharmacology. We have successfully utilised this strategy todiscover novel cysteine-containing peptides in various amphibianskin secretions. This led directly to the elucidation of thefull primary structure of several novel peptide bio-moleculesthat fit into different peptide families [3, 4]. It should be clearthat the approach is equally valid for peptide samples of anybiological origin.Being very generic, the method can be expanded — mutatismutandi — to look for analytes carrying many more PTMs, andwe, therefore designated the technique as PTM driven differentialpeptide display [3]. This is just limited by the creativityof the peptide chemist designing the specific chemical treatmentto be performed on the sample between the two LC-MSruns that will be compared.References1 Kastin, A.J. (2006) Handbook of Biologically ActivePeptides; Academic Press, 1640 pp.2 Verhaert, P. et al. (2009) Amphibian skin as a unique modelfor peptide analysis; NPC Highlights 9, 12-15.3 Evaristo, G.P.C. et al. (2012) PTM-driven DifferentialPeptide Display: Survey of Peptides Containing Inter/IntramolecularDisulfide Bridges in Frog Venoms, J. Proteomics:http://dx.doi.org/10.1016/j.prot.2012.09.0014 Evaristo, G. et al. (2012) The chains of the heterodimericamphibian skin antimicrobial peptide, distinctin, areencoded by separate messenger RNAs, J. Proteomics:http://dx.doi.org/10.1016/j.prot.2012.09.16Research teamFrom left to right:Geisa Evaristo, Peter Verhaert, Martijn Pinkse andMervin Pieterse, Department of Biotechnology, Facultyof Applied Sciences, Delft University of Technology.| 11ContactProf. Peter VerhaertAnalytical Biotechnology GroupDepartment of BiotechnologyDelft University of TechnologyJulianalaan 672628 BC Delft, the NetherlandsT +31 15 278 2332p.d.e.m.verhaert@tudelft.nlsummaryWe use liquid chromatography – tandem mass spectrometry(LC-MS/MS) to identify peptides with potential bioactivity incomplex mixtures of native peptides from natural sources. Asde novo sequencing of non-tryptic peptides is still a very laboriousand time-consuming task, it makes sense to recognisethe most promising peptides from the mixture first, to avoidspending unnecessary efforts in the energy demanding structuralanalysis of uninteresting sample components.The concept is that via a two-dimensional display of a (set of)LC-MS/MS run(s) various interesting features of the peptide sampleinvestigated are elegantly visualised. Against the backgroundof the sample’s complexity, a select set of typical structural characteristicsindicative for biological activity are highlighted. Someof these can already be seen directly from the straightforwardtwo-dimensional plot of LC-MS/MS retention time versus massto-chargeratio. Others reveal themselves only after selectivechemical treatment of the peptide sample, i.e. in a differentialtwo-dimensional display of the LC-MS/MS data of the treated anduntreated sample. Employing this strategy, we efficiently localisespecific post-translational modifications typical for various classesof bioactive peptides found in nature. Examples include thetargeting of carboxyterminally amidated peptides, and (singly ormultiply) disulfide bridge-linked peptides in the skin secretion offive different amphibians. This has already led to the discoveryand full primary structure elucidation of several novel peptidebiomolecules with interesting bioactivities.

Arjen Scholten, Christian Preisinger and Albert HeckInvestigating perturbationsinduced by kinase inhibitorGleevec12 | NPC Highlights 16 | November 2012The effect of Imatinib (Gleevec) on tyrosine kinases in Bcr-Ablpositive chronic myeloid leukaemia cells was followed for thefirst time using a new screening method. The researchers useda combination of stable isotope dimethyl labelling and peptidecentric immune-precipitation for targeted quantitative massspectrometry on tyrosine phosphorylated residues. Theirresults, as published recently in the journal Leukemia*,reveal new targets in the Bcr-Abl signalling network forintervention.The human genome encodes for 90 tyrosine kinases and 107tyrosine phosphatases. Mutation, overexpression, or functionalalteration of these enzymes is involved in many diseasesincluding cancer and immunodeficiency diseases. Therefore,targeted analysis of tyrosine phosphorylated residues on proteinsand elucidation of their biological role is mandatory forunderstanding their contribution to signalling networks and,consequently, to pathological processes.In the last few years, mass spectrometry (MS) has emerged asthe supreme tool for studying and characterising phosphorylationevents. However, due to the low stoichiometry of proteinphosphorylation, methods such as low-pH SCX, TiO 2 and IMACare used for the enrichment of phosphopeptides from complexproteolytic lysates.Adding yet another layer of difficulty, tyrosine phosphorylation* Preisinger, C et al. (2012) Imatinib-dependent tyrosine phosphorylation profilingof Bcr-Abl-positive chronic myeloid leukaemia cells, Leukemia, advance onlinepublication, 14 September 2012; doi: 10.1038/leu.2012.243is estimated to represent only about 1-2 percent of all humanphosphorylation events with respect to the more frequently occurringphosphorylations on serine (88 percent) or threonine (10percent) residues. Therefore, these aforementioned approachesare not well suited for the study of tyrosine phosphorylation.Immune affinity purification Several tools have been exploredfor the specific analysis of tyrosine phosphorylation.Profiling the global tyrosine phosphorylation state of cells canbe done using Immobilised Src homology 2 (SH2) domains,which bind selectively to specific tyrosine phosphorylatedsites. However, this approach is biased towards those phosphotyrosineproteins that interact with the SH2-containing bait.The development of specific, high affinity antibodies againstphosphorylated tyrosines provides an interesting alternativefor the global analysis of tyrosine phosphorylation [1]. Theseantibodies are obtained mostly by immunisation of a suitableorganism (e.g. mouse) with phosphotyramine or phosphotyrosinebound to carrier proteins. Traditionally, these antibodieshave been used to detect tyrosine phosphorylated residues ina protein of interest by Western blotting. Other applicationsinclude enzyme-linked immunosorbent assay (ELISA), immunohistochemisty,cell sorting by flow cytometry and immuno-

What this research is about:Advanced tools for in depth research onleukaemia drug effectChronic myeloid leukaemia is characterised by an abnormalincrease in immature white blood cells and it is treated withthe first line drug Imatinib, also known as Gleevec. The drugsuppresses the cell growth and division by inhibiting theresponsible kinase (Bcr-Abl) enzyme that is continuouslyactive due to an acquired mutation. “There is a lot ofknowledge about the drug and how it works on this kinase inthe leukemic cells, but what happens afterwards in the cellis not completely clear,” says Arjen Scholten, assistant professorof the Biomolecular Mass Spectrometry & Protemicsgroup of Utrecht University. “This kinase enzyme bindsspecifically with the amino acid tyrosine and attaches aphosphate label to it. It was not easy till now to detect thisphosphate modified tyrosine using conventional methods,since only a small percentage of all phosphate modificationspresent in the cell are of this specific type.”In this article the authors describe a method that canscreen specifically for these modified tyrosine substratesin leukemic cells. “We show that this tool can give insightinto a whole protein network at the same time. We identifiedapproximately two hundred modified tyrosine proteins,some already known and many new ones,” Scholten says.He picked one of the newly found proteins to study in moredepth in order to demonstrate the potential of this discoverytool.Since patients develop resistance to Imatinib after a certainperiod, other targets in the field of the kinase activity couldbe alternative approaches to circumvent resistance. Scholtenexplains: “Our studied protein might be an interesting targetfor a new drug. But that is long term thinking and not reallyour expertise. We used leukemic cells as a model system togain new insights into how Imatinib influences related proteinsin the leukemic cells. This screening technique can beenormously valuable for understanding biological networks.”| 13NPC E2: Chemical Approaches to Proteome Biologyfluorescence. Over the last few years, these antibodies havealso been used for the enrichment of tyrosine phosphorylatedproteins followed by mass spectrometry analysis [2]. However,detection of the actual phosphotyrosine residue is then hamperedby the high presence of unmodified peptides derivedfrom the captured phosphotyrosine modified protein and itsco-purifying binding partners.More recently, the field successfully shifted to the peptidelevel for the immune affinity purification (IAP) of phosphotyrosinepeptides from samples as complex as whole cell lysatedigests [3-5]. Most notably, Rikova et al. applied such an approachto investigate tyrosine kinase signalling across 41 lungcancer cell lines and over 150 tumours [5]. The most importantdifference with the protein level immunoprecipitation isthat this approach allows the identification and localisationof the exact phosphotyrosine residue in the protein on everyidentified protein.Here we describe how we adopted the IAP method to introducea robust and simple procedure that allows the identificationof hundreds of tyrosine phosphorylated residueswithin only 6 hours of mass spectrometry analysis time.When coupled to labelling strategies such as iTRAQ, SILAC ordimethylation (used here), this technique is able to extractbiologically rich quantitative information on tyrosine phosphorylationdriven signalling in differential conditions.Leukaemia and Gleevec The fusion kinase Bcr-Abl is the majorcause and pathogenetic principle of chronic myeloid leukaemia(CML). Bcr-Abl results from a chromosomal translocationthat fuses the bcr and the abl genes, thereby generating aconstitutively active tyrosine kinase, which stimulates severalsignalling networks required for proliferation and survival.Bcr-Abl’s oncogenic properties comprise both a kinase and ascaffold protein. A number of Bcr-Abl interaction partners anddownstream effectors have been described, improving ourunderstanding of the signalling networks deranged in CML. The‘core-interactome of Bcr-Abl’ entails seven major interactionpartners: GRB2, Shc1, Crk-l, c-Cbl, p85, Sts-1, and SHIP2 [6](see Figure 2).The introduction of the Bcr-Abl tyrosine kinase inhibitor (TKI)Imatinib (Gleevec) has been a landmark in the treatment ofCML. However, the development of Imatinib resistance poseschallenges to the clinical management of CML, and althoughsecond generation TKIs can block many Imatinib resistantmutants, they are ineffective against the common T315Imutation. An alternative strategy is to circumvent Imatinib

esistance by targeting downstream pathways essential fortransformation. Therefore, it is important to fully understandthese pathways. Large-scale (phospho)proteomics experimentshave addressed the phosphoproteome of Bcr-Abl positivecells, leading to the identification of an impressive number ofserine/threonine-phosphorylated sites (e.g. [7]). Typically, asmentioned above tyrosine-phosphorylation events remainedunderrepresented in these efforts. Since Bcr-Abl is itself atyrosine kinase and many of the protein-protein interactions inits network are dependent on tyrosine phosphorylation as well[6], we started out to map these.Enrichments Therefore we enriched, identified and quantifiedphosphotyrosine peptides by mass spectrometry [4] in order toexamine the effect of Imatinib on the CML blast crisis cell lineK562. First, the phosphorylation status and stability of severalkey proteins involved in Bcr-Abl signalling were evaluated inresponse to Imatinib (see Figure 1A). We treated cells withImatinib at concentrations of 0, 1, and 10 µM for 4 hours. Celllysates were subsequently digested with the proteases Lys-Cand trypsin, followed by stable isotope dimethyl labellingof the resulting peptides. The tri-plexed labelling approachallowed us to distinguish three peptide pools (light, intermediateand heavy), which were mixed in equal concentrations.Subsequently, tyrosine phosphorylated peptides were enrichedby immunoprecipitation and analysed by LC-MS. Analysis of thequantitative changes in tyrosine phosphorylation yielded 201unique quantifiable phosphotyrosine peptide triplets belonging14 | NPC Highlights 16 | Arjen Scholtento 141 proteins, by far exceeding all previous reports [7]. Ofthese, 87 peptides showed at least a 2-fold down-regulationafter treatment with Imatinib (see Figure 1B).Down-regulated Imatinib significantly decreased the tyrosinephosphorylation of many peptides originating from Bcr-Abland its core interactors (c-Cbl, CrkL and SHIP-2, see Figure1B and 2A). Furthermore, several proteins that have beenshown to play pivotal roles in Bcr-Abl dependent signalling(Gab1, Gab2, Shc1, Crk, ERK-2, STAT5A/B and Yes) displayedreduced tyrosine phosphorylation, often on multiple sites.Also, Src family kinase substrates exhibited reduced tyrosinephosphorylation, e.g. Cortactin, Catenin delta-1, nPKC-deltaand Paxillin. Finally, Imatinib reduced the tyrosine phosphorylationof several proteins involved in cytoskeletal regulation,such as MEMO1, Intersectin-2, Catenin delta-1, HEPL, GRF-1,Centaurin delta 2, and Plakophilin, which have not previouslybeen linked to Bcr-Abl signalling.A motif analysis on the sequences of significantly downregulatedtyrosine phosphorylated sites revealed a distinctenriched motif, YxxP, which is also recognised by Bcr-Abl. Intotal, 80 percent (23 out of 29) of the peptides harbouring thisphosphorylation motif were significantly down-regulated uponImatinib treatment. The YxxP motif resembles a classic bindingsite for SH2-domains and a consensus target sequence forphosphorylation by Bcr-Abl [8]. Peptides found in our screencontaining this motif include Bcr-Abl (Tyr115 and 128), STAT5Aand B (Tyr682 and 699, respectively), several sites in the adaptorproteins Gab1 (Tyr259, 373, and 406) and Gab2 (Tyr266,and 409) and all four phosphotyrosine containing peptides ofthe docking protein HEPL (Tyr174, 195, 244, and 329), indicatingthat the latter may represent a new Bcr-Abl substrate (seeFigure 1B).Several members of the Bcr-Abl core proteome [6] are adaptor/scaffoldproteins required for the generation of SH2- orPTB-domain binding sites that lead to tightly regulated proteininteractions [9]. Several of these peptides also contain theconsensus sequence YxxP of the enriched phosphorylationmotif. As these proteins bind to Bcr-Abl, the known and novelFigure 1 | Exploring the effect on tyrosine phosphorylation upon Imatinibinhibition of K562 cells.A. Overview of the quantitative proteomics workflow. Cells were treatedwith different doses of Imatinib (0, 1 and 10 µM) for 4 hours, followed bycell lysis and protein digestion. The peptides from each Imatinib treatmentwere then differentially labelled using stable isotope dimethyl labelling. Thethree differentially labelled digests were combined, followed by simultaneousenrichment of tyrosine phosphorylated peptides using immobilisedphosphotyrosine specific antibodies. The enriched fraction was analysed byLC-MS and changes in tyrosine phosphorylation quantified.B. Quantitative profiles of site-specific tyrosine phosphorylation upon Imatinibinhibition of Bcr-Abl. The changes in tyrosine phosphorylation versus thecontrol are represented on a log scale, marked in red, phosphorylation sitescontaining the YxxP motif.

Figure 2 | Alterations in the Gab2 interactome after Imatinib treatment.A. Imatinib treatment of K562 cells leads to destruction of the core Bcr-Abl complex (green, adapted from Brehme et al. [6]). A small selectionof peripheral interactors are depicted in blue. GAB2 (yellow) is used asflag-tagged entry point for interaction analysis upon Imatinib treatment.LC-MS/MS revealed release of GAB2 from the Bcr-Abl core proteome, exceptGRB2 (bold red). Dashed lines indicate as yet to be explored lost/intactinteractions within the Bcr-Abl Core proteome upon Imatinib treatment.B. Mutation of all regulated GAB2 tyrosine phosphorylated residues tophenylalanine reveals abolishment of cell periphery localisation of both GAB2and likely also the Bcr-Abl Core interactome.phosphorylation sites detected here may also be putative Bcr-Abl substrates.Gab2 interactome One member of the Bcr-Abl core proteome,the small adaptor protein GRB2, plays a key role inBcr-Abl signalling through binding to phosphorylated Tyr177on Bcr-Abl. This is required for activation of the Raf-MEK-ERKpathway in leukemic cells [10]. GRB2 also interacts with thedocking proteins Gab1 and Gab2 via GRB2’s SH3 domain andthe proline rich regions in both Gab proteins. The observedtyrosine phosphorylations of both Gab1 and Gab2 were founddown-regulated by Imatinib. We therefore quantitativelyevaluated the changes in the Gab2 interactome in responseto Imatinib treatment. Parallel FLAG-immuno-precipitationsfrom K562 cells transfected with either an empty FLAG-vectoror a FLAG-Gab2 encoding construct and treated with orwithout 10 µM Imatinib were performed. This affinity basedapproach revealed a severe alteration in the Gab2 interactomeafter Imatinib treatment (see Figure 2A). Whereas GRB2remained tightly bound, interactions between Gab2 and othercomponents of the Bcr-Abl core-interactome (e.g. Bcr-Abl,SHIP-2, and Shc1) were severely disrupted, suggesting that theGab2 interactome is dependent on the tyrosine phosphorylationstate of this protein. Subsequent immunoprecipitation ofendogenous Gab2 and Western blot detection of interactorsconfirms these results.The intracellular localisation of Gab2 and the closely relatedGab1 protein were evaluated by confocal microscopy. Whilewild-type GFP-Gab2 smoothly stained the circumference ofthe cell (see Figure 2B), the phospho-dead mutant where allImatinib sensitive phosphotyrosine residues were mutated tophenylalanine showed a severe alteration in localisation withspeckled staining of the cell periphery in addition to cytosolicstaining. Thus, tyrosine phosphorylation of Gab1 and Gab2 andthe subsequent ability to promote the formation of a complexwith Bcr-Abl and other core members appears to be requiredfor appropriate membrane localisation. These observationsstrongly advocate an involvement of Gab2 in the assembly ofthe Bcr-Abl signalling network at the cell periphery and theability of the core proteome to transform cells.summaryReferences1 Hunter, T. (1998) The Croonian Lecture 1997. The phosphorylationof proteins on tyrosine: its role in cell growth anddisease. Philos Trans R Soc Lond B Biol Sci 353, 583-605.2 Salomon, A. R. et al. (2003) Profiling of tyrosine phosphorylationpathways in human cells using mass spectrometry.Proc Natl Acad Sci U S A 100, 443-448.3 Rush, J. et al. (2005) Immunoaffinity profiling of tyrosinephosphorylation in cancer cells. Nat Biotechnol 23, 94-101.4 Boersema, P. J. et al. (2010) In-depth qualitative andquantitative profiling of tyrosine phosphorylation using acombination of phosphopeptide immunoaffinity purificationand stable isotope dimethyl labeling. Mol Cell Proteomics9, 84-99.5 Rikova, K. et al. (2007) Global survey of phosphotyrosinesignaling identifies oncogenic kinases in lung cancer. Cell131, 1190-1203.6 Brehme, M. et al. (2009).Charting the molecular networkof the drug target Bcr-Abl. Proc Natl Acad Sci U S A 106,7414-7419.7 Pan, C. et al. (2009) Global effects of kinase inhibitors onsignaling networks revealed by quantitative phosphoproteomics.Mol Cell Proteomics 8, 2796-2808.8 Amanchy, R. et al. (2007) A curated compendium of phosphorylationmotifs. Nature biotechnology 25, 285-286.9 Pawson, T. (2004) Specificity in signal transduction: fromphosphotyrosine-SH2 domain interactions to complex cellularsystems. Cell 116, 191-203.10 Pendergast, A. M. et al. (1993) BCR-ABL-induced oncogenesisis mediated by direct interaction with the SH2 domainof the GRB-2 adaptor protein. Cell 75, 175-185.ContactArjen Scholten, PhDBiomolecular Mass Spectrometry & Proteomics groupUtrecht UniversityPadualaan 83584 CH Utrecht, the NetherlandsT +31 30 253 6793a.scholten@uu.nl| 15To probe the effect of Imatinib on tyrosine kinase Bcr-Abl positive cells, we applied a targeted quantitativemass spectrometry based proteomics approach. We usedphosphotyrosine specific immuno-capturing to systematicallyscreen for Imatinib sensitive tyrosine phosphorylation eventsin K562 cells. Our data reveal that several core Bcr-Ablinteractors are putative direct substrates, but also newputative targets are discovered. In following up a novelImatinib inhibited target, the adaptor protein Gab2 wasfound to regulate localisation of the Bcr-Abl core complex tothe cell periphery in a phosphorylation dependent manner,enabling us to add crucial new dimensions of detail to the Bcr-Abl signalling network. This work provides insights into howprotein phosphorylation influences protein networks and showswhich widespread effects a kinase inhibitor may have on thenetwork biology of this leukemic cell.

Serena Di Palma and Shabaz MohammedAccelerating towardsultrasensitive proteomeanalysis16 | NPC Highlights 16 | November 2012One of the difficulties with current proteomics analysismethods is the need for samples of hundreds ofthousands or millions of cells. In many cases, this amountof cells is not available, for example analysing adultstem cells, tumour biopsy or samples obtained by lasermicrodissection. A new 2D approach using hydrophilicseparation as the first step gives excellent resolution andallows for more sensitive proteome analysis.In the last decade, the field of proteomics has rapidly progressedand elicited considerable interest. Continuous technologicaladvances in mass spectrometry (MS) instrumentation,peptide fractionation strategies and bioinformatics have led toincreasing knowledge of proteomes. However, improvementsare still needed, especially for the investigation of limitedamounts of material such as laser microdissected tissues orspecific subsets of cells obtained through flow cytometry [1]. Asuite of special analytical tools is required to deal with underrepresented(or limited) samples and to further the single-cellsingle-proteome dream.We believe that a new peptide separation strategy based onhydrophilic interaction liquid chromatography (HILIC) willhelp to accelerate proteomics towards a high level of depth inregard to sensitivity, proteome coverage and comprehensivity.Here we highlight how HILIC can be implemented into shotgunproteomics, to find applications in situations where limitedsample availability represents a challenge.Advantages of HILIC HILIC is a relatively new player in theproteomics field, providing particular advantages in comparisonto more established techniques such as reversed-phase(RP) and ion-exchange (IEX) chromatography [2]. First, thehigh organic composition of the buffer, with low salt concentrationor volatile salts, makes HILIC especially suitable forelectrospray ionisation, potentially increasing its sensitivity.Second, the special affinity for polar compounds allows theanalysis of highly hydrophilic species that would otherwisebe lost under RP analysis. Third, the high orthogonality orcomplementarity to RP makes HILIC an ideal candidate inmultidimensional approaches for the analysis of complexpeptide mixtures, representing an excellent alternative tostrong-cation exchange (SCX) as a first dimension [3]. All thesereasons have contributed to an exponential increase in theapplication of HILIC chromatography in proteomics, especiallyfor the analysis of polar protein post translation modifications(PTMs) such as glycosylation and phosphorylation [4].

What this research is about:Just a few thousand cells neededfor high quality analysisAnalysing just a single cell among thousands of others is theholy grail of proteomics nowadays. It would certainly help indiagnosing cancer, for example. The study of cell-to-cell communicationwould also greatly benefit from such an approach.Current proteomics research requires hundreds of thousandsor even millions of cells per sample. Common methods forpeptides analysis using mass spectrometry need these amountsto accurately identify large numbers of peptides.Serena Di Palma spent her PhD study finding new peptideseparation strategies and, more importantly, miniaturisingthose systems, with the aim of developing analytical methodsthat use small sample sizes. She replaced the commonlyused chromatographic separation method known as SCX(strong-cation exchange) with a different chromatographicmethod only recently introduced in proteomics called HILIC(hydrophilic interaction liquid chromatography). HILICseparates peptides mainly by hydrophilicity and to a lesserextent by charge. “This means that peptides do not elute inbig clumps (clusters of similar charge) but more separated,”says Di Palma. Separating hydrophilic peptides has becomemore important in the last years because the interest ofproteomics has shifted towards analysis of post-translationalmodifications of proteins. These are usually polar modificationssuch as glycosylation and phosphorylation, generatingsamples with a large amount of hydrophilic peptides.Di Palma designed a miniaturised set-up using a zwitterionicHILIC, as the peptide fractionation step, combined withreversed-phase (RP) chromatography in a 2D system. “Havingzwitterionic stationary phases means that the total chargeof the material will remain zero, which gives a specialadvantage for analyses performed at different pH values,”says Di Palma. She compared her results to a traditional proteomicsanalysis and found that many more proteins couldbe identified using the HILIC separation step before applyingthe RP chromatography. The protein identification matchedresults from large scale proteomics analysis. The researchersthink that this method can be very useful for analysing smallsamples obtained by laser microdissection or flow cytometry.| 17NPC E1: NPC New T2: Separation Proteome and Biology Enrichment of Plants Tools in ProteomicsIn our laboratory, we successfully designed a two-dimensional(2D) system based on the combination of zwitterionic HILICand RP, both in nanoscale format [5]. One of the major issuesassociated with the combination of HILIC and RP arises fromthe incompatibility of the mobile phases when employing anon-line setup. To overcome this problem, we refined and miniaturisedour HILIC system, as schematically shown in Figure 1,employing column dimensions as small as 75 µm in the internaldiameter. During the ZIC-cHILIC elution, fractions are directlycollected into a 96-well plate already containing an acidifiedwater solvent. The HILIC fractions are then sufficiently aqueousand compatible with the subsequent RP analysis, withoutfurther manipulation. This strategy could also be defined asin-line, as it is optimised to reduce sample handling, whichFigure 1 | Schematic of the 2D system based on a ZIC-cHILIC fractionation, followed by RP-MS analysis.

Figure 2 | Schematic representation of the employed workflow for theisolation of adult colon stem cells from mouse intestine and the subsequentproteomics analysis performed by the 2D HILIC-RP or 1D RP strategy.causes sample loss and contamination. We showed that this 2Dapproach combines excellent resolution in both the first andsecond dimension. Furthermore, it allows for more sensitiveproteome analysis in comparison to current methods, suchas SCX, that require more input material. In fact, when weanalysed prototypical cancer cell lines (HeLa) employing amere few micrograms of starting material, we could achieveproteome coverage comparable to ‘large scale’ strategies.Application to stem cells The identification and characterisationof downstream Wnt target genes led to the discovery of18 | NPC Highlights 16Lgr5, a gene that is uniquely expressed in the stem cells ofseveral adult tissues such as intestine, hair follicles and stomach.A breakthrough in the study of this gene has been thegeneration of a mouse strain in which GFP has been knockedinto the Lgr5 locus. Moreover, a cell sorting method basedon FACS has been established that allows a high enrichment(>95%) of these GFP-marked adult colon stem cells, albeitin rather small quantities for proteomics studies. To achievea targeted proteomics analysis at an ultrasensitive level,optimised methods are required. These include a combinationof specific sample preparation for maximal recovery withsensitive methods for separation, detection and identificationof all possible peptides/proteins. Thus, we probed thesensitivity of our new 2D technology with a real small-scalesample scenario, and analysed FACS-obtained colon stem cells,directly after tissue extraction [6]. A schematic workflow issummarised in Figure 2.A population of 30,000 colon stem cells was FACS sorted andsubsequently digested using Lys-C and Trypsin. We started witha total of 30,000 cells to have sufficient material for a systematicevaluation using one-dimensional (1D) and two-dimensional(2D) separations. A portion of the sample correspondingto 5,000 cells was subjected to a standard 1D RP nanoLC-MSanalysis using a rather long five hour gradient elution. Thisallowed the identification of 380 proteins and 759 unique peptides,in line with our expectation. Afterwards, we applied our2D HILIC-RP strategy to the analysis of 10,000 colon stem cellsand compared it with the 1D RP-MS analysis. The HILIC fractionationled to 27 fractions, wherein half of each fraction wasfurther analysed by RP-MS. Effectively, considering the overallworkflow, we analysed the same amount for both 1D and 2Dapproaches, consisting of 5,000 stem cells. Combining all thedata from the 2D strategy allowed the cumulative identificationof 15,775 unique peptides, originating from 3,775 proteins(see Figure 3). Within each fraction, we reported the numberof unique peptides and also evaluated the distribution ofpeptide charge (2+, 3+, 4+ and 5+), as indicated in Figure 3a.Although 2+, 3+ and 4+ charged peptides eluted over quite anumber of fractions, a trend of an increase in peptide chargein the later fractions was observed.A valid alternative to current methods Comparing this resultwith the 1D analysis, we reached a 20-fold and 10-fold improvementin peptide and protein identifications, respectively.The proteome coverage we achieved at the time rivalled thatreached by current state-of-the-art proteome analyses thatoften require several millions of cells. A detailed comparisonbetween the two strategies revealed that only five (out of380) proteins present in the 1D analysis were not observed inthe two-dimensional ZIC-cHILIC-RP experiment, illustratingthat very little sample loss occurs incorporating the additional| Serena Di PalmaFigure 3 | Analysis of FACS-sorted colon stem cells with a 2D ZIC-cHILIC approachand a 1D RP strategy. (A) The cumulative number of identified uniquepeptides and proteins and distribution of unique peptides (including peptidecharge) across the HILIC fractions and the 1D RP run.Venn diagrams showing the overlap of identified (B) unique peptides and (C)protein between the 2D (reported as combined fractions) and 1D approach.

separation step (see Figure 3c). In addition, only 101 peptides(corresponding to approximately 0.6% of the total of 15,775peptides) found in the 1D analysis were not identified in the2D experiment, which potentially may be attributed to theincompatibility of certain peptides with the HILIC solvents. Ingeneral, both Venn diagrams (see Figure 3b and 3c) showedlarge overlaps, suggesting that the peptides identified in the1D RP analysis are merely higher abundant peptides that areall also detected in the ZIC-cHILIC-RP strategy.A further comparison between the obtained proteome dataand previous microarray experiments from the same colonstem cells confirmed the quality of our method, showing that95% of the proteins detected in this study were also foundto be expressed at the mRNA level. Furthermore, 21% of theGFP+ colon stem cell specific genes could be detected in ourproteomics screen, and two of the identified proteins, CD44and EphB3, have been reported to be expressed highest at thebottom of the crypt, where the colon stem cells reside.A Gene Ontology analysis for molecular function and biologicalprocesses of the 3,775 detected proteins revealed that the 2Dstrategy covered all the most important functional categories,including, for instance, proteins involved in transcriptionmechanisms, in receptor activity and in translation regulationactivity.Major step forward Notwithstanding the huge advancesmade in proteomics in the last decade, sensitive technologiesare still needed to access proteome-wide data for a limitednumber of cells, for instance originating from laser microdissections,flow cytometry, tumour biopsy and other scenarioswhere sample material will be a limiting factor. We demonstratedthat the 2D HILIC based strategy developed in ourlaboratory leads to a significant reduction of sample complexitywith nearly negligible sample loss. The approach describedhere represents a major step forward towards a more sensitiveproteome analysis. We propose to introduce ZIC-cHILIC as afractionation method in shotgun proteomics strategy for theanalysis of only a few thousands of cells. [7] Furthermore, thisstrategy, in combination with metabolic or chemical labellingbased quantification [8], can investigate and compare withhigh-sensitivity specific cell proteomes.References1 Altelaar A.F.M. et al. (2012) Trends in ultrasensitive proteomics.Current Opinion in Chemical Biology 16, 206-213.2 Di Palma S. et al. (2012) Recent advances in peptideseparation by multidimensional liquid chromatography forproteome analysis. Journal of Proteomics 75, 3791-3813.3 Boersema P.J. et al. (2007) Evaluation and optimization ofZIC-HILIC-RP as an alternative MudPIT strategy. Journal ofProteome Research 6, 937-946.4 Boersema P.J. et al. (2008) Hydrophilic interaction liquidchromatography (HILIC) in proteomics. Anal Bioanal Chem391, 151-159.5 Di Palma S. et al. (2011) Zwitterionic hydrophilic interactionliquid chromatography (ZIC-HILIC and ZIC-cHILIC)provide high resolution separation and increase sensitivityin proteome analysis. Anal Chem 83, 3440-3447.6 Di Palma S. et al. (2011) Highly sensitive proteome analysisof FACS-sorted adult colon stem cells. J Proteome Res 10,3814-3819.7 Di Palma S. et al. (2012) ZIC-cHILIC as a fractionationmethod for sensitive and powerful shotgun proteomics. NatProtocols 7, 2041–2055.8 Di Palma S. et al. (2011) Evaluation of the deuteriumisotope effect in zwitterionic hydrophilic interaction liquidchromatography separations for implementation in a quantitativeproteomic approach. Anal Chem 83, 8352-8356.ContactSerena Di Palma, MScBiomolecular Mass Spectrometry and Proteomics GroupUtrecht UniversityPadualaan 83584 CH Utrecht, The NetherlandsT +31 30 253 3664S.DiPalma@uu.nl| 19summaryIn proteomics, multidimensional liquid chromatographycombined with mass spectrometry has become a standardtechnique to reduce sample complexity and tackle the vastdynamic range. Such fractionation is necessary to obtaina comprehensive analysis of biological samples such astissues and cell lines. However, extensive fractionationcomes at the expense of sample loss, hampering the analysisof limited material. Here we describe a highly sensitivetwo-dimensional chromatographic strategy based on acombination of hydrophilic interaction liquid chromatography(with a zwitterionic stationary phase) and reversed-phasechromatography. This strategy allows shotgun proteomicsanalysis with minimal sample loss. We applied this technologyto the analysis of a limited number of FACS-sorted colon stemcells extracted from mouse intestine, obtaining proteomecoverage comparable to current methods that generallyrequire 100-fold more starting material. We propose that thisalternative multidimensional chromatographic technology willfind ample application in biomedical and biological research,such as in the analysis of distinct cellular populations obtainedby laser microdissection or flow cytometry.

Teck Yew Low, Flore Kruiswijk, Roberto Magliozzi andDaniele GuardavaccaroGenotoxic stress affectsprotein synthesis throughregulation of eEF2K20 | NPC Highlights 16 | November 2012Eukaryotic elongation factor 2 kinase (eEF2K) is a crucialenzyme in repressing translation elongation. We have recentlydiscovered this protein as a substrate of the SCF βTrCP ubiquitinligase using a combined mass spectrometry-based proteininteraction screening with an in-house developed technique forphosphopeptide analysis. Focusing on the biological functionsof eEF2K, we investigated how the enzyme is controlled bygenotoxic stress and how this leads to a decrease in translationelongation and protein synthesis.Cells activate surveillance molecular networks known as DNAdamage checkpoints to protect their genome from environmentaland metabolic genotoxic stress. Depending on the type andextent of DNA lesions and the cellular context, damaged cellswith an activated checkpoint can undergo senescence, die byapoptotic cell death, or repair the damaged genome and, aftercheckpoint termination, resume their physiological functions.Genotoxic stress has a greater effect on gene expression at thelevel of mRNA translation than at the level of transcription,probably because protein synthesis requires about 40% of thetotal cellular energy, and cells need to couple stress responsesto metabolic demands. Therefore, in response to genotoxicstress, cells aim to preserve energy by reducing protein synthesisin order to be able to repair the damage.Despite the major impact of genotoxic stress on mRNA translation,no information is available on how translation elongationis affected by genotoxic stress, and there have been few studiesdirected toward understanding the regulation of protein synthesisby the DNA damage response.Translation elongation Mammalian target of rapamycin (mTOR)is a serine/threonine protein kinase that integrates growth andstress signals and promotes protein synthesis. The mTOR pathwaypromotes translation elongation by inhibiting eukaryoticelongation factor 2 kinase (eEF2K), which phosphorylates andinactivates eEF2, a factor that mediates the translocation stepof peptide-chain elongation. The activity of eEF2K is controlledunder various conditions. For example, stimuli that induceprotein synthesis trigger the inactivation of eEF2K and the subsequentdephosphorylation of eEF2. In contrast, deficiencies innutrients or energy lead to activation of eEF2K and impairmentof translation elongation.In addition to this level of regulation, eEF2K abundance iscontrolled by the ubiquitin-proteasome system. In this systemsubstrates are tagged by covalent attachment of multipleubiquitin molecules and then the polyubiquitylated proteinsare degraded by the 26S proteasome. Several enzymes areinvolved in this system, but the E3 ubiquitin ligases representthe essential regulators of ubiquitylation because they physicallyinteract with target substrates. Although it is known that

What this research is about:How the ubiquitin-proteasome systemcontrols fundamental cellular functions“Daniele Guardavaccaro started as a new group leader at the Hubrecht Institute in 2008 and came to us with a veryinteresting biological question,” says Teck Yew Low, researcher at the NPC and Biomolecular Mass Spectrometryand Proteomics group (Utrecht University) and highly experienced in new techniques for analysing proteins.“Guardavaccaro wanted to know which proteins bind to specific enzymes, known as E3 ubiquitin ligases.” Theseenzymes tag proteins that need to be destroyed by attaching a small protein called ubiquitin to them. Next, a complexcalled proteasome seeks out and destroys all molecules with the ubiquitin tag. By finding proteins bound to E3,one may discover new targets that are marked for destruction by the ubiquitin-proteasome system.Guardavaccaro, Low and colleagues identified many novel E3 binding proteins. One of them, an enzyme knownas eEF2K, is a key repressor of mRNA translation — the process by which mRNAs are translated into proteins. Theresearchers zoomed in on eEF2K and found important details on how mRNA translation is regulated. Translation isa process that requires a considerable amount of energy. “We found that when the cellular DNA is damaged, forexample upon exposure to chemical mutagens, the repressor of mRNA translation (eEF2K) is rapidly activated andtranslation is temporarily slowed down. As a consequence, a large amount of energy is saved and used by the cellto fix the damaged DNA. Once the damaged DNA is repaired, the repressor of translation is first tagged by ubiquitinand then destroyed by the proteasome. As a result, mRNA translation is resumed,” explains Low.In this research, Low and colleagues also carried out extensive analysis of proteins modified by phosphate groups(phosphoproteins). “Regulation of proteins by phosphorylation is the prevalent mode of regulation of protein functionand its study is essential in our research. We frequently apply a number of techniques recently developed inour lab to enrich for phosphoproteins and facilitate their analysis by mass spectrometry.” Low and Guardavaccaroare currently continuing their collaboration to analyse other E3 binding proteins.| 21NPC T1: Cancer ProteomicsFigure 1 | The structure of the SCF βTrCP ubiquitin ligase and its phosphorylation-dependentinteraction with the substrate. The F-box protein βTrCPis the substrate receptor subunit that recruits specific substrate proteins.Through its WD40 β-propeller structure, βTrCP recognises a diphosphorylatedmotif with the consensus DpSGXX(X)pS in which the serine residues are phosphorylatedto allow interaction with βTrCP.eEF2K abundance is controlled by the ubiquitin-proteasomesystem [1], the biological importance of the ubiquitylation anddegradation of eEF2K and the identity of the E3 ubiquitin ligaseinvolved are unknown.Proteomics screening To identify substrates of the ubiquitinligase SCF βTrCP (see Figure 1), we used immuno-affinity chromatographyfollowed by tandem mass spectrometry (MS/MS) [2].First, we expressed FLAG–hemagglutinin (HA) epitope–taggedβTrCP2 (FLAG-HA-βTrCP2) in human embryonic kidney HEK293Tcells and analysed by MS proteins that co-purified with FLAG-HA-βTrCP2 after sequential FLAG and HA immunoprecipitations.We identified nine unique peptides derived from eEF2K [3,4].Subsequently, by reciprocal immunoprecipitation with FLAG-HA-eEF2K, we recovered peptides corresponding to βTrCP1 andβTrCP2. This is not surprising since βTrCP1 and βTrCP2 shareidentical biochemical properties and substrates.The WD40 β-propeller structure of βTrCP binds its substrates(like eEF2K) through a diphosphorylated motif (phosphodegron)with the consensus DpSGXX(X)pS. The generation of severaleEF2K mutants with replacements of residues within this motifshowed that the motif surrounding Ser441 and Ser445 is highlyconserved and required for the binding of eEF2K to βTrCP (seeFigure 2A and 2B). Furthermore, we also showed that phosphorylationis required for the interaction of eEF2K with βTrCP. AneEF2K peptide containing phosphorylated Ser441 and Ser445 in-

Figure 2 | A. The βTrCP-eEF2K interaction depends on a conservedphosphodegron. Ser441 and Ser445 in eEF2K are required for the associationwith βTrCP1. Top: schematic representation of four putative eEF2Kphosphodegrons. Bottom: HEK293T cells were transfected with FLAG-taggedβTrCP1 and with either an empty vector, HA-tagged eEF2K wild type (WT),or mutants, treated with MG132, and lysed. Whole-cell extracts weresubjected to either immunoblotting or immunoprecipitation with anti-FLAGresin and subsequent immunoblotting. The eEF2K(S441A/S445A) mutantimmunoprecipitated less with βTrCP1 compared to wild-type eEF2K and theother mutants.B. Alignment of the amino acid regions corresponding to the βTrCP-bindingmotif (highlighted in yellow) in previously reported βTrCP substrates and eEF2Korthologs (top). Amino acid sequence of the eEF2K double mutant is shown(bottom).teracted with βTrCP1, but the corresponding nonphosphorylatedpeptide was unable to do so. To analyse the phosphorylation ofthe degron in vivo, we used an in-house developed techniquebased on Ti(4+)-immobilised metal ion affinity chromatography(Ti-IMAC) as described [5] in combination with orbitrap massspectrometry.22 | NPC Highlights 16 | Teck Yew LowTo test whether SCFβTrCP was directly responsible for thepolyubiquitylation of eEF2K, we reconstituted the ubiquitylationof eEF2K in vitro. Immunopurified βTrCP1, but not an inactiveβTrCP1 mutant, induced the in vitro ubiquitylation of eEF2K.Crucial inhibitor Since eEF2K is a crucial inhibitor of translationelongation and because inhibition of protein synthesisis a common response to stress conditions, we examined theabundance of eEF2K in response to genotoxic stress. U2OScells were synchronised in the G2 phase of the cell cycle andthen treated with a low dose of doxorubicin to activate theG2 DNA damage checkpoint. Subsequently, cells were allowedto recover from the DNA damage checkpoint after extensivewashing in a drug-free medium. The abundance of eEF2Ksteadily decreased during checkpoint silencing. A similar patternof changes in eEF2K abundance was observed in experimentsin which ionising radiation was used.Moreover, eEF2K degradation was inhibited when βTrCP expressionwas silenced in U2OS cells during checkpoint silencing.Using a phosphospecific antibody that recognises eEF2K onlywhen it is phosphorylated on Ser441 and Ser445, we were ableto show that phosphorylation of the eEF2K degron is inducedby DNA damage in G2 and preceded degradation of eEF2K.Together, these findings imply that βTrCP targets eEF2K fordegradation upon checkpoint silencing.The kinase eEF2K controls translation elongation by phosphorylatingeEF2 on Thr56, thereby inactivating it. The affinity ofeEF2 for the ribosome is reduced when Thr56 is phosphorylatedand therefore the ribosome-eEF2 complex is not formed. Ourresults indicate that phosphorylation of eEF2 on Thr56 increasesupon checkpoint activation and decreases during checkpoint silencing.Increased phosphorylation of eEF2 on Thr56 in responseto DNA damage is mediated by eEF2K. This was confirmed byour experiment in which eEF2K expression was silenced in U2OScells. The cells were synchronised in G2 and then pulsed withdoxorubicin to induce DNA damage; eEF2K knockdown blockedthe DNA damage–induced phosphorylation of eEF2 on Thr56.The kinase eEF2K is phosphorylated on Ser398 by AMPK (adenosinemonophosphate-activated protein kinase), a phosphorylationevent that leads to eEF2K activation. AMPK is activated bythe products of two p53 target genes, Sestrin1 and Sestrin2, inresponse to genotoxic stress. Using an antibody that specificallyrecognises eEF2K phosphorylated on Ser398, we showed thatin response to genotoxic stress, Ser398 was phosphorylated.Inhibition of AMPK prevented the increase in eEF2K phosphorylationon Ser398 and eEF2 phosphorylation on Thr56 in responseto genotoxic stress.Together, these data indicate that genotoxic stress in G2 cellsstimulates AMPK, which phosphorylates eEF2K on Ser398, causingits activation. In turn, activated eEF2K phosphorylates eEF2on Thr56, blocking its activity.Checkpoint initiation Checkpoint silencingAMPKAMPK+SCF βTrCPeEF2KeEF2KeEF2eEF2Translation elongation Translation elongationFigure 3 | Model of eEF2K regulation in response to genotoxic stress. Bluelines represent activated proteins, and red indicates inactive or degradedproteins.

Resuming protein synthesis Since eEF2K has autophosphorylationactivity [6], we hypothesised that eEF2K, once activated,may autophosphorylate its degron, thus triggering the bindingof eEF2K to βTrCP. Indeed, immunopurified wild type eEF2Kcould autophosphorylate on Ser441 and Ser445 and bind toβTrCP in vitro whereas a kinase dead eEF2K mutant could not.We then wanted to know whether βTrCP-mediated degradationof eEF2K is required to resume protein synthesis uponcheckpoint silencing. Cells expressing the degradation-resistantform of eEF2K that were exposed to genotoxic stress displayeda slower decrease in phosphorylation of eEF2 on Thr56 duringsilencing of the DNA damage checkpoint. These cells alsodisplayed a decreased rate of global protein synthesis duringcheckpoint silencing when compared to control cells.Initiation and silencing In this article we show that uponactivation of the DNA damage checkpoint, AMPK mediates theactivation of eEF2K, which in turn phosphorylates eEF2, leadingto a decrease in translation elongation rates. Subsequently,eEF2K autophosphorylation generates a phosphodegron for therecruitment of the SCF βTrCP ubiquitin ligase. This event triggersthe ubiquitylation of eEF2K and its proteasome-mediateddegradation, which releases the inhibitory effect on eEF2 andtranslation elongation (see Figure 3).Regulation of translation elongation in response to DNA damagemight have several advantages over controlling translationinitiation. Indeed, elongation inhibition during checkpoint activationavoids the disassembly of polysomes, which, by stallingribosomes on the mRNAs, might allow for mRNA protection fromdegradation or sequestration into stress granules. This mechanismwill also ensure that translation can rapidly resume whenthe checkpoint is turned off.SCF βTrCP is implicated in the degradation of several substratesduring the recovery from DNA damage and replication stresscheckpoints. Indeed, βTrCP is required to reactivate the cyclindependentkinase Cdk1 by targeting the Cdk1 inhibitors Claspinand Wee1 for proteasome-dependent degradation and turn offthe DNA repair machinery by causing the destruction of theFanconi anemia protein FANCM. Our findings show that βTrCPis also needed during checkpoint silencing to resume proteinsynthesis by triggering proteolysis of eEF2K, suggesting that theubiquitin ligase SCF βTrCP coordinates different processes (suchas cell cycle progression, DNA repair, and protein synthesis) thatare critical for checkpoint termination.References1 Arora, S. (2005) , Identification of the ubiquitin-proteasomepathway in the regulation of the stability of eukaryoticelongation factor-2 kinase. Cancer Res 65, 3806–3810.2 Low, T.Y. et al. (2012) Unraveling the ubiquitin-regulatedsignaling networks by mass spectrometry-based proteomics.Proteomics Sep 28. doi: 10.1002/pmic.201200244.[Epub ahead of print]3 Kruiswijk, F. et al. (2012) Coupled activation and degradationof eEF2K regulates protein synthesis in response togenotoxic stress. Sci Signal, 5(227):ra40.4 Meloche, S and Roux, P.P. (2012) F-box proteins elongatetranslation during stress recovery. Sci Signal, 5(227):pe25.5 Zhou, H. et al. (2011) Enhancing the identification ofphosphopeptides from putative basophilic kinase substratesusing Ti (IV) based IMAC enrichment. Mol Cell Proteomics10 (10):M110.006452.6 Diggle, T.A. et al. (1999) Analysis of the domain structureof elongation factor-2 kinase by mutagenesis. FEBS Lett,457(2):189-92.Research team| 23From left to right: Teck YewLow from the BiomolecularMass Spectrometry andContactProteomics Group, Utrecht;Teck Yew Low, PhDDaniele Guardavaccaro,Biomolecular Mass SpectrometryFlore Kruiswijk andand Proteomics GroupRoberto Magliozzi fromKruytgebouw Z605KNAW Hubrecht Institute,Padualaan 8Utrecht3584 CH Utrecht, the NetherlandsT +31 30 253 9403t.y.low@uu.nlsummaryThe kinase eEF2K controls the rate of peptide chain elongationby phosphorylating eEF2, the protein that mediates themovement of the ribosome along the mRNA by promotingtranslocation of the transfer RNA from the A to the Psite in the ribosome. eEF2K-mediated phosphorylation ofeEF2 on threonine 56 (Thr56) decreases its affinity for theribosome, thereby inhibiting elongation. Here we show thatin response to genotoxic stress, eEF2K was activated byAMPK (adenosine monophosphate–activated protein kinase)–mediated phosphorylation on serine 398. Activated eEF2Kphosphorylated eEF2 and induced a temporary ribosomalslowdown at the stage of elongation. Subsequently, duringDNA damage checkpoint silencing, a process required to allowcell cycle reentry, eEF2K was degraded by the ubiquitinproteasomesystem through the ubiquitin ligase SCF βTrCP(Skp1–Cul1–F-box protein, β-transducin repeat–containingprotein) to enable rapid resumption of translation elongation.This event required autophosphorylation of eEF2K on acanonical βTrCP- binding domain. The inability to degradeeEF2K during checkpoint silencing caused sustainedphosphorylation of eEF2 on Thr56 and delayed the resumptionof translation elongation. Our study therefore establishesa link between DNA damage signalling and translationelongation.

Blighted by KenningApples infected with knowledgeA collaboration between the NetherlandsProteomics Centre and Charlotte Jarvis24 |From November 15 th to December 6 th 2012 the bio-artproject Blighted by Kenning is showcased at Hôtel Droog,Amsterdam. The Netherlands Proteomics Centre (NPC)and the British artist Charlotte Jarvis worked for almost ayear on the project, in which apples grown at the Haguewere ‘contaminated’ with synthetic DNA encoded with theUniversal Declaration of Human Rights. Martje Ebberink,communications manager at the NPC, tells us more aboutthe realisation of the project, the collaboration betweenCharlotte Jarvis and the scientists, and the hurdles theyhad to overcome.Martje Ebberink remembers it as if it were yesterday. Sheexplains: “The 6 th of December 2011 was the turning pointfor Blighted by Kenning. Although remarkably enough, it wasalso the day we lost the Designers & Artists for GenomicsAwards (DA4GA) contest. This inventive competition bringstogether elements from life sciences, art and design with theaim of bridging the gap between the life sciences and creativeindustries.” During the so-called ‘speed dates’ scientists fromleading genomics institutes, artists and designers were introducedto each other. Genomics centres and artists that werea ‘match’ were then asked to submit a proposal. On the lastday of the contest, seven finalists presented their collaborativeideas to a professional jury from the fields of art andscience. “From the moment we met Charlotte, we knew thiscould be the start of an innovative relationship. We stronglybelieve in Charlotte and the project,” states Ebberink. “Afterall the enthusiasm and positive reactions from professionalsin the arts and sciences, we were disappointed that we werenot one of the prize winners. We certainly would have beenpleased to bring the project to life with a budget of €25.000and showcase it at the museum Naturalis in Leiden.” The NPCfound Charlotte’s idea so inspiring that they decided to makeit a reality without the help of the DA4GA, and named herresident artist. From then on things moved very quickly.Artistically and scientifically uniqueAfter much preparation, Charlotte presented her vision at theNPC Progress Meeting in February 2012. Scientists at the NPCthen went to work on bio-engineereing a bacteria so that TheUniversal Declaration of Human Rights would be encoded intoits DNA. The synthetic DNA was extracted from the bacteriaOn August 4, 2012 the grand opening of the Blighted by Kenningexhibit was held in a former dairy warehouse on the coast of Suffolk(UK) where a small apple orchard of thirteen trees was installed.

During the opening ceremony Charlotte Jarvis ate one of the forbidden fruits.and used to ‘contaminate’ apples from The Hague, which weresent to genomics laboratories around the globe. Researcherswho received the contaminated apples were asked to sequencethe DNA, find the hidden message and send back atranslation.Life sciences join forces with art & designThe project is essentially a performance art piece in which anidea is biologically and literally spread across the world. It isartistically and scientifically unique in that it will create aninternational network of genomics institutes and a meaningfulmessage by spreading a genetically engineered material. “Ourhope is that Blighted by Kenning will contribute to the understandingof how information is encoded and expressed withinDNA. We want to encourage people to think about what kindof information is contained within genetic material and howthis can be manipulated. By creating a network of internationalinstitutions that contribute to the project, a statementwill be made about the importance of genomics research andthe interaction between science, technology and society,”says Ebberink.Getting the licenceWhile working on the project, Charlotte Jarvis and the NPCencountered several obstacles. Ebberink explains: “At first wewanted to use the bacteria itself to contaminate the apples,not just its DNA. After consulting scientists at the NPC, somecompanies and a science museum, it was anticipated that itwas nearly impossible to legally exhibit a Genetically ModifiedOrganism (GMO) in a gallery.” Reinout Raijmakers, ManagingDirector of the Bijvoet Centre of Utrecht University, whoserved as a consultant for the scientific part of the project,came up with the idea to extract the DNA from the bacteria(leaving ‘naked DNA’ which does not carry the environmentalrisks associated with GMOs) and use this inert material tocontaminate the apples. “Using naked DNA allowed us morefreedom that might not otherwise be possible. For instance,we wanted to be able to ask people to eat the apples. Eatingthe apple allows scientists to make a gesture in support ofgenetics research,” Ebberink argues.The decision was made to use the naked DNA, but things tooklonger than expected to get started. The most difficult partof the project was getting the licence to make the bacteria.She explains: “It turns out that we were the first group inthe Netherlands to suggest encoding a fully synthetic proteinsequence in DNA. Consequently, we were required to provideadditional safety measures in order to obtain a license tomake the DNA sequence.” After a couple of months, the NPCfinally received notice that the permit was approved, and theapples were produced and mailed off to different corners ofthe earth.Orchard inside the galleryOn August 4, 2012 the grand opening of the Blighted byKenning exhibit was held in a former dairy warehouse on thecoast of Suffolk (UK) where a small apple orchard of thirteentrees was installed. Ebberink reports: “Hanging on oneof the trees was a sample of our forbidden fruit. There wasalso a billboard set up in the space, displaying images of TheDeclaration of Human Rights expressed as a protein. Films ofthe scientists eating the fruit, documents sent by institutesafter sequencing the apples and a wall of correspondencedetailing the making of the project were included in the exhibit.”During the opening ceremony Charlotte Jarvis ate oneof the forbidden fruits. The next installment of the exhibit atHotel Droog in Amsterdam will be more or less the same, butwill include a documentary of the project.Round table events were organised at both locations to questionthe current state of play with regards to popular perceptionsof genetics. The topics were introduced by a number ofspeakers from science, the press and the arts, with an openfloor discussion to close out the debate.| 25Images of The Declaration of Human Rights expressed as a protein.Join us at Hôtel DroogBlighted By Kenning is on view till December 6th atHôtel Droog, Staalstraat 7B, 1011 JJ Amsterdam.For more information, visitwww.drooglab.com/projects/events/ orwww.netherlandsproteomicscentre.nl

ValorisationNPC Valorisation Voucher in practiceMaximum exploitationrequires clear agreementsResearchers at Utrecht University and PamGene solvedthe problem of detecting phosphatase activity with a newpeptide microarray. With the NPC Valorisation Voucher theywere able to make the step towards a commercial product.“The success is chiefly based on the fruitful cooperationbetween university and company,” assert the researchersinvolved.In November 2011 Jeroen van Ameijde came up with a luminousidea, started a collaboration with PamGene and was also awardedan NPC Valorisation Voucher. One year later we talked toJeroen van Ameide and Rob Ruijtenbeek, who look back on theeffective progress of their commercialisation of a microarraytechnology for proteomics. “Now the validation phase has beensuccessfully finished, we plan to offer the assay to a first batchof clients,” they say. “That is a big advantage of working witha company that has a network of prospective clients,” explainsOne year ago the 6th NPC Valorisation Voucher had been awarded to (fromleft to right) Rob Ruijtenbeek (PamGene), Rob Liskamp (UU) and Jeroen vanAmeijde (UU).26 | NPC Highlights 16 | November 2012Jeroen Van Ameijde from the Medicinal Chemistry and ChemicalBiology group at Utrecht University. “We as university researcherswill help solve unexpected hurdles, but it is up to PamGeneto commercialise the assay further.”Van Ameijde is very pleased with the successful collaboration.“Our groups have worked closely together for a long time.Publishing scientific results, which can be a controversialissue, has never been a problem since we have clear agreementswith PamGene. So if relevant, the patent can be filedon time. But it is an issue that is always there,” Van Ameijdeadds. “When you regulate this topic by setting terms for publishingand filing patents at the start of a new collaborationthere shouldn’t be a problem at all,” he advises.distinguish from other molecules in the cell because it haslost its phosphate label.”Together with Rob Ruijtenbeek of PamGene, the company thatproduces microarray assays for functional readouts of kinaseand nuclear receptor activities, Van Ameijde had a luminousidea. What if they added an extra label to the substratemolecule, which would give a detectable product when thephosphatase is active. “It was a risky idea,” he says. “Innature things work very specifically. So if you add even a smallchemical group to a molecule, it can lose its interaction withthe enzyme, especially when the label is near the place wherephosphatases are active. To our surprise we saw it did notbother them at all.”Discovery and developmentKinases and phosphatases play a key role in signal transductionpathways in the cell. Together they regulate the phosphorylationpatterns by respectively attaching and removingphosphate groups to and from proteins. “How the kinaseenzyme family works and how we can intervene with drugshas been a subject of research for decades,” explains vanAmeijde. “There are very good tools available for studyingand profiling kinase activity. Similar assays for researchingphosphatases are still lacking.” Knowledge about both enzymefamilies is important, since even a single disruption inphosphorylation patterns can cause a wide range of diseases,such as cancer. Van Ameijde: “The problem with measuringphosphatase activity is that the resulting molecule is hard toTowards a real productIn the first test series they used about eight substrates. For areal product, dozens if not hundreds of substrates on the spotsof a microarray are needed to make valuable phosphataseprofiles. Van Ameijde: “We used the NPC Valorisation Voucherto scale it up and to demonstrate its effectiveness even incomplex biological samples, and thereby take a step towards areal product.” They also tested different situations that mightbe interesting for future customers. For instance, a companythat has developed a new drug to inhibit a certain phosphatasewould like to know how selective it is and whether itblocks other important enzymes too. “That is a huge problemwith kinase inhibitors and has attracted the most attention.These kinds of tools are therefore extremely essential.”

Novel biomarkers patent filednpc valorisationArzu Umar, NPC project leader for breast cancer proteomics at Erasmus MC, has foundnovel biomarkers for determining the prognosis of triple negative breast cancer. Theprognostic profile helps defining the group of patients that are cured by surgery and preventsunnecessary treatment with chemotherapy. In addition, these biomarkers may serve astargets for development of novel therapies for triple negative breast cancer. A patent hasrecently been filed.No prognostic protein markers are currently available in theclinic for triple negative breast cancer. The newly developedprotein profile has been generated with a dedicated state-ofthe-artproteomics platform and provides in-depth proteomeanalysis of minute amounts of clinical sample. The profilehas recently been validated in an independent multi-centrepatient cohort using the same proteome analysis. At present,quantitative, targeted mass spectrometry assays are beingdeveloped that should enable translation to the clinic.Triple negativeBreast cancer affects one in eight women throughout theirlifetimes and accounts for about 485,000 deaths annually. Thesenumbers will increase by 13%, up to 548,000 incidences in 2019(Datamonitor 2010). Most breast cancers are ER, PR or HER2positive and can be effectively treated with targeted therapiesdirected against these proteins, such as hormonal therapiesblocking production or function of estrogens, and antibody orkinase inhibitor therapies blocking the HER2 pathway. A minority,10-15%, of breast cancers lack the three important clinicalmarkers ER, PR and HER2 and are classified as triple negative(TN). Patients with TN breast cancer have an overall poor prognosisdue to the aggressive nature of these tumours and lack ofsuitable targets for therapy. As a consequence, patients receiveRon Heeren co-founder ofOmics2Image B.V.Ron HeerenThe start-up company Omics2Imagehas recently been established tobring technological innovations fromthe AMOLF group, lead by Prof. RonHeeren, to the market. The firstproduct of the start-up is the IonPixcamera. This camera in combinationwith a microscope mode massspectrometry system providesextremely high spatial resolution molecular images and hasthe capability of capturing several ion masses in one measurementcycle. The AMOLF technostarter Omics2Image worksclosely with Amsterdam Scientific Instruments, which focuseson other areas of detector technology. Omics2Image is the 3rdhigh-tech spin-off under the aegis of holding company ParticlePhysics Inside Products (P2IP). P2IP helps to navigate seed andearly stage companies through the valorisation process, i.e.feasibility studies and business development.NPC Valorisation Voucher forHuib Ovaa and Sjaak NeefjesNPC theme leader Huib Ovaa and NPC project leader SjaakNeefjes (Netherlands Cancer Institute) have been awarded anNPC Valorisation Voucher for their proposal ‘Identification ofthe methotrexome for drug development’. Methotrexate is anagent used in cancer therapy, but is also effective in the treatmentof rheumatoid arthritis and other autoimmune diseases.Ovaa and Neefjes are exploring a new target that explainsthe effects of methotrexate on autoimmunity, and chemicalproteomics plays a central role in their approach. They aim todevelop better medication without the side effects associatedwith methotrexate treatment. With this valorisation voucherthe Neefjes and Ovaa labs target novel agents for rheumatoidarthritis therapy to be further developed in a novel startupcompany.Arzu Umarharsh chemotherapy that may display severe side effects andmay be very debilitating. Of all patients with TN breast cancer,only 25% will develop distant metastases within five years aftersurgery. Therefore, the TN breast cancer can be divided intoa good and poor prognostic group. However, as there is no effectiveprognostic screening method, patients with long-termprogression free survival are also receiving harsh chemotherapy.Predicting beforehand which patients have favourable diseaseoutcome and thus do not have to be treated with harsh chemotherapywould greatly improve the quality of life for thesepatients and reduce health care costs. In addition, markers forpoor prognosis could be used as new targets against which newtherapies for TN breast cancer can be developed.Call for interested partiesA patent application has been filed and Erasmus MC is lookingfor a partner for a license to the technology with potentialresearch collaboration. Interested parties are cordially invitedto contact the Erasmus MC TTO to investigate the opportunitiesfor licensing and/or research collaboration.Contact information: Louise Hoppel, Business Developer,Technology Transfer Office, Erasmus MC, Rotterdam.(l.hoppel@erasmusmc.nl / +31 10 704 3343)| 27

Dutch Techcentre for the Life SciencesTop-end research hotel for integrative biologyThe Dutch Techcentre for Life Sciences (DTL) wasintroduced in the spring issue of NPC Highlights. Now, sixmonths down the road, DTL’s interim managing director(and managing director of the Netherlands BioinformaticsCentre NBIC) Ruben Kok provides an update. “The conceptof the NPC Research Hotels is a key element in the set-upof DTL.”Ruben KokFirst, a quick recap for those who are unfamiliar with thestructure of DTL and what it envisions. Driven by the increasinglycomplex nature of life sciences research, a number ofadvanced technology centres and platforms in the Netherlandsare building a new collaborative environment: the DutchTechcentre for Life Sciences. DTL offers scientists (in30 | NPC Highlights 16 | November 2012academia, medical centres and industry) the opportunity totackle questions that require the use of multiple technologies,without having to address the different technology platformsseparately. Ruben Kok elucidates: “Biologists increasingly lookfor ways to tackle the complexity of living systems on a newlevel. What they need is an integrative biology approach,where cross-links are established between technology disciplines.With DTL, we are creating an interface between thescientist and the various technology platforms and infrastructuresfor integrating and analysing big data.”Cross-technology researchTo date, DTL covers genomics/next generation sequencing,proteomics, metabolomics, advanced bioimaging as experimentaltechnologies, systems biology, e-science and bioinformaticson the data handling and modelling side. It is nosurprise that the Netherlands Proteomics Centre is the‘proteomics-supplier’ of DTL. But there is more. NPC wasthe first to pioneer the concept of research hotels, and thissuccessful concept will be continued at DTL. “When lookingat the structure of DTL, most participating centres can beviewed as ‘research hotels’, local facilities that focus on aspecific discipline, such as proteomics or bio-imaging. We arenow building a network in which these individual hotels areconnected to each other to create a cross-technology researchfacility. The ultimate goal is to create an environment wherea biologist can present a research question or experiment thatrequires the use of multiple technologies and he or she is subsequentlyoffered high-class expertise and facilities, like in atop-end hotel, to perform those experiments ‘in one go’. The‘guest’ only has to address one ‘desk’, that being DTL.”Big Data infrastructureSignificantly, the DTL collaboration does not stop when thedata are generated. A key element of DTL is DISC, whichstands for Data Integration and Stewardship Centre. Kok elaborates:“Each DTL facility is connected in DISC to ascertainthe best integration and handing of the experimental data andother sources of relevant information. All the different technologiesrelating to data processing, storage and analysis cometogether here to form a broadly accessible Big Data infrastructure.”DISC is also the Dutch ‘node’ in ELIXIR, the EuropeanLife Sciences Infrastructure for Biological Information. “Beingassociated with international initiatives like ELIXIR is crucialfor the future of life sciences research in the Netherlands,”says Kok, “that is why collaborating in entities like DTL isimportant for individual groups and technology centres. Ifwe want to benefit from international programmes and, evenbetter, influence the set-up and priorities of such initiatives,we have to be able to make a strong case. And that requirescritical mass, as the participants in DTL clearly recognise.” Heemphasises that DTL is open to all kinds of parties, whethertheir focus is on drug development, bio-based materials,equipment manufacturing, food, computational science — thelist of potential topics is virtually endless. Kok concludes:“DTL welcomes anyone who operates at the forefront of lifesciences.”More information on DTL and DISC: www.dtls.nl

NPC Supervisory BoardThe NPC Supervisory Board represents the highest ‘levelof control’. The board advises the Executive Board onall matters related to the overall strategy and has toapprove its main decisions. The Supervisory Board hasfinal responsibility for all NPC activities. The SupervisoryBoard meets twice a year, with additional meetings ifnecessary. Marcel Wubbolts (DSM) and René Medema(Netherlands Cancer Institute) both joined the NPCSupervisory Board last year. They explain the importanceof proteomics in current life sciences research.Proteomics in the development of sustainable production“Sustainability is a leading principle in managing our projects,”says Marcel Wubbolts, Chief Technology Officer (CTO) atDSM and member of the NPC Supervisory Board. Wubbolts isresponsible for overall technology for DSM and that includesthe development of sustainable production methods forpharma, nutritional and materials applications. Wubboltsexplains: “Our products include biobased materials for cars,biofuels from non-food feedstock and enzymes for medicineproduction. Sustainable production lowers energy and materialuse enormously. For example, initially the production ofCephalexin, a semi-synthetic cephalosporin, from Penicillin Grequired thirteen steps. We can now do it in three steps: firstfermentation using a fungus in which engineered genes wereintroduced, followed by two enzymatic steps, all in water. Theuse of material and energy was reduced by 65 percent.”Wubbolts stresses that proteomics techniques are essentialfor diverse applications such as quality control of productionstrains and enzymes. “When you introduce new genesinto a fungus to improve a production process, you need toknow if the enzymes reachthe proper location withinthe fungus and whetherthey perform the way theyshould. That the fungusmakes the desired productalso needs to be proved. Sowe use techniques like massspectrometry, NMR, 2-D gelelectrophoresis and crystallography.”Marcel WubboltsCollaboration with universities and institutes such as NPCplays an important role in the product development of DSM.“The initial phase of trying a new technology is often riskyand costly. Working in consortiums divides the risks and costs.When we ascertain that a technique is strategically importantvia such a partnership, we also build it up in house. Wehave learned a great deal from universities this way and willcontinue to do so.”| 31Proteomics in oncobiology“Proteomics plays a very important role in oncology,” affirmsProf. René Medema, chairman of the Board of the NetherlandsCancer Institute (NKI-AVL) and member of the NPC SupervisoryBoard. “Advances in both genomics and proteomics will enablethe transition to personalised medicine. Thanks to these omicstechnologies, the diversity amongst tumours can be discerned.Proteomics enables us to discover biomarkers with which onecan follow tumours, for example to detect whether theyrespond to a certain therapy or not. Proteomics is indispensableas a tool to unravel the fundamentals of oncobiology.”Medema, who is also member of the NPC Supervisory board,is very glad to have access to new proteomics techniquesthrough the NPC. “As a hospital one could decide to developall the proteomics techniques in-house, but this would requireenormous investments in people and equipment. However, asa user of the technologies you do want to have access to thelatest innovations, so we arevery glad that the NPC connectsus to research groups that wantto bring these technologies toan even higher level.”Medema stresses that theright infrastructure has provenvital to the innovative cancerresearch with which the NKIhas built its internationalreputation. The improvementRené Medemaof cancer therapies and effectiveuse of available medication have also profited from thisinfrastructure. Thanks to the NPC, proteomics research in theNetherlands is ideally organised, whereby developments arequickly passed on to other researchers.”

Top publications with NPC contributionIn this NPC HighLights we provide a short list ofpapers that appeared recently in some of the topjournals and to which NPC participants contributed.With the guarantee of being by far notcomprehensive, this overview shows some elegantground-breaking research.Tumour suppressor RNF43 is a stem-cellE3 ligase that induces endocytosis of WntreceptorsKoo, B.K., Spit, M., Jordens, I., Low, T.Y., Stange, D.E.,van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J.,Maurice, M.M., Clevers, H.Nature (2012) Aug 30;488(7413):665-9. doi: 10.1038/nature11308.Hubrecht Institute, KNAW and University Medical CenterUtrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.LGR5+ stem cells reside at crypt bottoms, intermingled withPaneth cells that provide Wnt, Notch and epidermal growth32 | NPC factor Highlights signals. Here 16 we | November find that the 2012 related RNF43 and ZNRF3transmembrane E3 ubiquitin ligases are uniquely expressedin LGR5+ stem cells. Simultaneous deletion of the two genesencoding these proteins in the intestinal epithelium of miceinduces rapidly growing adenomas containing high numbersof Paneth and LGR5+ stem cells. In vitro, growth of organoidsderived from these adenomas is arrested when Wnt secretionis inhibited, indicating a dependence of the adenomastem cells on Wnt produced by adenoma Paneth cells. In theHEK293T human cancer cell line, expression of RNF43 blocksWnt responses and targets surface-expressed frizzled receptorsto lysosomes. In the RNF43-mutant colorectal cancercell line HCT116, reconstitution of RNF43 expression removesits response to exogenous Wnt. We conclude that RNF43 andZNRF3 reduce Wnt signals by selectively ubiquitinating frizzledreceptors, thereby targeting these Wnt receptors for degradation.The landscape of cancer genes andmutational processes in breast cancerStephens, P.J. et. AlNature (2012) May 16;486(7403):400-4. doi: 10.1038/nature11017.Cancer Genome Project, Wellcome Trust Sanger Institute,Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK.All cancers carry somatic mutations in their genomes. A subset,known as driver mutations, confer clonal selective advantage oncancer cells and are causally implicated in oncogenesis, and theremainder are passenger mutations. The driver mutations andmutational processes operative in breast cancer have not yetbeen comprehensively explored. Here we examine the genomesof 100 tumours for somatic copy number changes and mutationsin the coding exons of protein-coding genes. The number ofsomatic mutations varied markedly between individual tumours.We found strong correlations between mutation number, age atwhich cancer was diagnosed and cancer histological grade, andobserved multiple mutational signatures, including one presentin about ten per cent of tumours characterized by numerousmutations of cytosine at TpC dinucleotides. Driver mutationswere identified in several new cancer genes including AKT2,ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1and TBX3. Among the 100 tumours, we found driver mutationsin at least 40 cancer genes and 73 different combinations ofmutated cancer genes. The results highlight the substantialgenetic diversity underlying this common disease.High-sensitivity Orbitrap mass analysis ofintact macromolecular assembliesRose, R.J., Damoc, E., Denisov, E., Makarov, A., Heck, A.J.Nature Methods (2012) Oct 14. doi: 10.1038/nmeth.2208.[Epub ahead of print]Biomolecular Mass Spectrometry and Proteomics, BijvoetCenter for Biomolecular Research and Utrecht Institute ofPharmaceutical Sciences, Utrecht University, Utrecht,The Netherlands. Netherlands Proteomics Center, Utrecht,The Netherlands.The analysis of intact protein assemblies in native-like statesby mass spectrometry offers a wealth of information on theirbiochemical and biophysical properties. Here we show thatthe Orbitrap mass analyzer can be used to measure proteinassemblies of molecular weights approaching one megadaltonwith sensitivity down to the detection of single ions. Minor instrumentalmodifications enabled the measurement of variousprotein assemblies with outstanding mass-spectral resolution.Protease bias in absolute protein quantitationPeng, M., Taouatas, N., Cappadona, S., van Breukelen, B.,Mohammed, S., Scholten, A., Heck, A.J.Nature Methods (2012) May 30;9(6):524-5. doi: 10.1038/nmeth.2031.Trypsin is the most commonly used protease in typical proteomicsexperiments. (Absolute) Quantitation using spectralcounts provides insight into protein abundances. This isdependent on the protease used. In other words, the use ofa single protease, such as trypsin, can introduce biases in theobserved protein abundances.

The life history of 21 breast cancersNik-Zainal, S., et al.Cell (2012) May 25;149(5):994-1007. Epub 2012 May 17.Cancer Genome Project, Wellcome Trust Sanger Institute,Hinxton CB10 1SA, UK.Cancer evolves dynamically as clonal expansions supersedeone another driven by shifting selective pressures, mutationalprocesses, and disrupted cancer genes. These processes markthe genome, such that a cancer’s life history is encrypted inthe somatic mutations present. We developed algorithms todecipher this narrative and applied them to 21 breast cancers.Mutational processes evolve across a cancer’s lifespan,with many emerging late but contributing extensive geneticvariation. Subclonal diversification is prominent, and mostmutations are found in just a fraction of tumor cells. Everytumor has a dominant subclonal lineage, representing morethan 50% of tumor cells. Minimal expansion of these subclonesoccurs until many hundreds to thousands of mutations haveaccumulated, implying the existence of long-lived, quiescentcell lineages capable of substantial proliferation upon acquisitionof enabling genomic changes. Expansion of the dominantsubclone to an appreciable mass may therefore represent thefinal rate-limiting step in a breast cancer’s development, triggeringdiagnosis.Triple bioorthogonal ligation strategy forsimultaneous labeling of multiple enzymaticactivitiesWillems, L.I., Li, N., Florea, B.I., Ruben, M., van der Marel,G.A., Overkleeft, H.S.Angew Chem (2012) Apr 27;51(18):4431-4. doi: 10.1002/anie.201200923. Epub 2012 Mar 21.Leiden Institute of Chemistry and Netherlands ProteomicsCentre, Gorlaeus Laboratories, Einsteinweg 55,2333 CC Leiden, The Netherlands.Chd proteins are ATP-dependent chromatin remodelingenzymes implicated in biological functions from transcriptionalelongation to control of pluripotency. Previous studiesof the Chd1 subclass of these proteins have implicated themin diverse roles in gene expression including functions duringinitiation, elongation, and termination. Furthermore, someevidence has suggested a role for Chd1 in replication-independenthistone exchange or assembly. Here, we examine rolesof Chd1 in replication-independent dynamics of histone H3in both Drosophila and yeast. We find evidence of a role forChd1 in H3 dynamics in both organisms. Using genome-wideChIP-on-chip analysis, we find that Chd1 influences histoneturnover at the 5’ and 3’ ends of genes, accelerating H3 replacementat the 5’ ends of genes while protecting the 3’ endsof genes from excessive H3 turnover. Although consistent witha direct role for Chd1 in exchange, these results may indicatethat Chd1 stabilizes nucleosomes perturbed by transcription.Curiously, we observe a strong effect of gene length on Chd1’seffects on H3 turnover. Finally, we show that Chd1 also affectshistone modification patterns over genes, likely as a consequenceof its effects on histone replacement. Taken together,our results emphasize a role for Chd1 in histone replacementin both budding yeast and Drosophila melanogaster, and surprisinglythey show that the major effects of Chd1 on turnoveroccur at the 3’ ends of genes.An N-terminal acidic region of Sgs1 interactswith Rpa70 and recruits Rad53 kinase tostalled forksHegnauer, A.M., Hustedt, N., Shimada, K., Pike, B.L., Vogel,M., Amsler, P., Rubin, S.M., van Leeuwen, F., Guénolé, A., vanAttikum, H., Thomä, N.H., Gasser, S.M.EMBO J. (2012) Jul 20;31(18):3768-83. doi: 10.1038/emboj.2012.195. Epub 2012 Jul 20.Friedrich Miescher Institute for Biomedical Research, Basel,Switzerland [2] Faculty of Sciences, University of Basel, Basel,Switzerland..| 33Three at the same time: A ligation strategy combining tetrazine-norbornenecycloaddition, Staudinger-Bertozzi ligation,and copper(I)-catalyzed click reaction was used to label thethree catalytic activities of the proteasome simultaneously ina single experiment. The orthogonality of the three ligationreactions enables selective monitoring of multiple targets atthe same time in complex biological samples.A key role for Chd1 in histone H3 dynamics atthe 3’ ends of long genes in yeast.Radman-Livaja, M., Quan, T.K., Valenzuela, L., Armstrong, J.A.,van Welsem, T., Kim, T., Lee, L.J., Buratowski, S.,van Leeuwen, F., Rando, O.J., Hartzog, G.A.PLoS Genet. (2012) Jul;8(7):e1002811. Epub 2012 Jul 12.Department of Biochemistry and Molecular Pharmacology,University of Massachusetts Medical School, Worcester,Massachusetts, USA.DNA replication fork stalling poses a major threat to genomestability. This is counteracted in part by the intra-S phasecheckpoint, which stabilizes arrested replication machinery,prevents cell-cycle progression and promotes DNA repair. Thecheckpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLMcontribute synergistically to fork maintenance on hydroxyurea(HU). Both enzymes interact with replication protein A (RPA).We identified and deleted the major interaction sites on Sgs1for Rpa70, generating a mutant called sgs1-r1. In contrast to ahelicase-dead mutant of Sgs1, sgs1-r1 did not significantly reducerecovery of DNA polymerase α at HU-arrested replicationforks. However, the Sgs1 R1 domain is a target of Mec1 kinase,deletion of which compromises Rad53 activation on HU. Fullactivation of Rad53 is achieved through phosphorylation of theSgs1 R1 domain by Mec1, which promotes Sgs1 binding to theFHA1 domain of Rad53 with high affinity. We propose that therecruitment of Rad53 by phosphorylated Sgs1 promotes thereplication checkpoint response on HU. Loss of the R1 domainincreases lethality selectively in cells lacking Mus81, Slx4, Slx5or Slx8.

34 |The Lgr5 intestinal stem cell signature:robust expression of proposed quiescent‘+4’ cell markersMuñoz, J., Stange, D.E., Schepers, A.G., van de Wetering, M.,Koo, B.K., Itzkovitz, S., Volckmann, R., Kung, K.S., Koster, J.,Radulescu, S., Myant, K., Versteeg, R., Sansom, O.J., van Es,J.H., Barker, N., van Oudenaarden, A., Mohammed, S., Heck,A.J., Clevers, H.EMBO J. 2012 Jun 12;31(14):3079-91. doi: 10.1038/emboj.2012.166.Bijvoet Center for Biomolecular Research, Utrecht Institute forPharmaceutical Sciences, University, The Netherlands.Two types of stem cells are currently defined in small intestinalcrypts: cycling crypt base columnar (CBC) cells and quiescent‘+4’ cells. Here, we combine transcriptomics with proteomicsto define a definitive molecular signature for Lgr5(+)CBC cells. Transcriptional profiling of FACS-sorted Lgr5(+)stem cells and their daughters using two microarray platformsrevealed an mRNA stem cell signature of 384 unique genes.Quantitative mass spectrometry on the same cell populationsidentified 278 proteins enriched in intestinal stem cells. ThemRNA and protein data sets showed a high level of correlationand a combined signature of 510 stem cell-enriched genes wasdefined. Spatial expression patterns were further characterizedby mRNA in-situ hybridization, revealing that approximatelyhalf of the genes were expressed in a gradient withhighest levels at the crypt bottom, while the other half wasexpressed uniquely in Lgr5(+)stem cells. Lineage tracing usinga newly established knock-in mouse for one of the signaturegenes, Smoc2, confirmed its stem cell specificity. Using thisresource, we find-and confirm by independent approaches-thatthe proposed quiescent/’+4’ stem cell markers Bmi1, Tert,Hopx and Lrig1 are robustly expressed in CBC cells.Other highlighted publicationsAlbers, H.M., Ovaa, H.Chemical evolution of autotaxin inhibitorsChem Rev. (2012) May 9;112(5):2593-603. Epub 2012 Feb 15.Kruiswijk, F., Yuniati, L., Magliozzi, R., Low, T.Y., Lim, R.,Bolder, R., Mohammed, S., Proud, C.G., Heck, A.J., Pagano,M., Guardavaccaro, D.Coupled activation and degradation of eEF2K regulates proteinsynthesis in response to genotoxic stressSci Signal. 2012 Jun 5;5(227):ra40.Meinema, A.C., Poolman, B., Veenhoff, L.M.The transport of integral membrane proteins across thenuclear pore complexNucleus. (2012) Jul 1;3(4).Ongay S., Boichenko A., Govorukhina N., Bischoff R.Glycopeptide enrichment and separation for proteinglycosylation analysisJ Sep Sci. (2012) Sep;35(18):2341-72. doi: 10.1002/jssc.201200434.Bischoff, R., and Schlüter, H.Amino acids: Chemistry, functionality and selected nonenzymaticpost-translational modificationsJ. Proteomics (2012) 75, 2275-2296Kovanich, D., Aye, T.T., Heck, A.J., Scholten, A.Probing the specificity of protein-protein interactions byquantitative chemical proteomicsMethods Mol Biol. (2012) 803:167-81.Kovanich, D, Cappadona, S., Raijmakers, R., Mohammed, S.,Scholten, A., Heck, A.J.Applications of stable isotope dimethyl labeling in quantitativeproteomicsAnal Bioanal Chem. (2012) Sep;404(4):991-1009.van Geel, R., Pruijn, G.J., van Delft, F.L., Boelens, W.C.Preventing thiol-yne addition improves the specificity ofstrain-promoted azide-alkyne cycloadditionBioconjug Chem. (2012) Mar 21;23(3):392-8. Epub 2012 Mar 8.Krijgsheld, P., Altelaar, A.F., Post, H., Ringrose, J.H., Müller,W.H., Heck, A.J., Wösten, H.A.Spatially Resolving the Secretome within the Mycelium of theCell Factory Aspergillus nigerJournal of Proteome Research (2012) 11 (5), 2807-2818Lindhoud, S., van den Berg, W.A., van den Heuvel, R.H., Heck,A.J., van Mierlo, C.P., van Berkel, W.J.Cofactor Binding Protects Flavodoxin against Oxidative StressPLoS One. 2012;7(7):e41363. (IF: 4.092)Smaczniak, C., Immink, R.G., Angenent, G.C., Kaufmann, K.Developmental and evolutionary diversity of plant MADSdomainfactors: insights from recent studiesDevelopment. (2012) Sep;139(17):3081-98. doi: 10.1242/dev.074674.Eschrich, S., Fulp, W.J., Pawitan, Y., Foekens, J.A., Smid, M.,Martens, J.W., Echevarria, M., Kamath, V., Lee, J.H., Harris,E.E., Bergh, J., Torres-Roca, J.F.Validation of a radiosensitivity molevular signature in breastcancerClin Cancer Res. (2012) Sep 15;18(18):5134-5143. Epub 2012Jul 25.Godinho, M.F., Wulfkuhle, J.D., Look, M.P., Sieuwerts, A.M.,Sleijfer, S., Foekens, J.A., Petricoin, E.F. 3rd, Dorssers, L.C.,van Agthoven, T.BCAR4 induces antiestrogen resistance but sensitizes breastcancer to lapatinibBr J Cancer. (2012) Sep 4;107(6):947-55. doi: 10.1038/bjc.2012.351. Epub 2012 Aug 14.Richau, K.H., Kaschani, F., Verdoes, M., Pansuriya, T.C.,Niessen, S., Stüber, K., Colby, T., Overkleeft, H.S., Bogyo, M.,Van der Hoorn, R.A.Subclassification and biochemical analysis of plant papain-likecysteine proteases displays subfamily-specific characteristics.Plant Physiol. 2012 Apr;158(4):1583-99. Epub 2012 Feb 27.

10 years Progress inProteomeBiology10 Years NPC: Progress in Proteome BiologyThe meeting is dedicated to the 10th anniversary of the NPC. In a special programmeoutstanding national and international scientists will present their views on the newestdevelopments in proteomics with lectures, NPC project posters and an exhibition. Thecelebration will be completed with the NPC Anniversary Dinner on February 11th.| 35We look forward to seeing you at this anniversary meeting!Registration & abstract deadline: 21 January 2013www.netherlandsproteomicscentre.nlprogressmeeting@npc.genomics.nlConfirmed speakers include Steven A. Carr Joshua Coon Steve HahnAlbert HeckConnie JimenezRoland KanaarJeroen KrijgsveldMadelon MauriceRené MedemaChristine MummeryMaurien OlsthoornBert PoolmanCarol RobinsonBen ScheresMarc TimmersMathias UhlenArzu Umar

upcoming events15 November-6 December20 November 201217 January 201311-12 February 20139-13 June 2013|||||Blighted by Kenning, Hôtel Droog, Amsterdam, The NetherlandsNGI Life Sciences Momentum, Fokker Terminal, The Hague, The NetherlandsNPC PhD day, DSM Biotechnology Center, Delft, The NetherlandsNPC Progress Meeting 2013, Media Plaza, Utrecht, The NetherlandsASMS Conference, Minneapolis Convention Center Minneapolis, MNGolden futureIn 2002, the Netherlands Genomics Initiative (NGI) waslaunched. Consequently, it was decided that the field ofgenomics could benefit from a solid infrastructure with acollaborative foundation. From this fundamental idea came theinitiative taken by Albert Heck and his partners to put togetherthe Netherlands Proteomics Centre (NPC) – a seamless andaward-winning fit, backed by an excellent proposal.When the first phase of the NPC began, in 2003, I had theprivilege of serving as chairman of the Supervisory Board, andI can now proudly say that I have enjoyed the position for tenyears. Fortunately, Albert Heck and his team have continuouslysucceeded in securing funds for a follow-up programme,starting with an award of 25 million euros, another estimating22 million euros, and finally 13.5 million euros for 2014-2018.To further substantiate that our work has had a significantimpact, knowledge institutes and the business community havecontinued to support the NPC by matching these funds.Eduard KlasenProfessor EmeritusDean & Member ExecutiveBoard LUMC till June 2012Chairman NPC ExecutiveBoard 2003-2012Proteomics is a crucial part of Dutch life sciences, with manyspin-offs, and thanks to the NPC, we can now say that theNetherlands is a major play in this field. For example, there arethe countless scientific publications, the well-attended yearlyprogress meetings, and let’s not forget the successful HUPO in2008 meeting which brought international protein researchersfrom all over the globe to the Netherlands.On a larger scale, proteomics will undoubtedly make a majorcontribution to the understanding of biological processes atthe protein level. This will lead to the development of newmedicines and biomarkers that will have a huge impact on thediagnosis and treatment of diseases, but it will also affect otherfields in the life sciences e.g. the agro-food sector.The solid network built up in the Netherlands is the resultof creativity and perseverance, but most notably the will tocooperate by sharing and delivering TOP results. Continuedexpansion in the coming years and the drive to lead activitiesin the Life Sciences and Health sector will prove to be highlyproductive for the Dutch life sciences, but most certainlyalso for the NPC. As chairman of the Supervisory Board I feelconfident in passing on the torch to my successor, and reflectfondly on my years of contribution. I would like to thank AlbertHeck, his team and partners, and also my fellow members ofthe Supervisory Board for a productive and pleasant workingexperience. I wish the NPC a golden future.16