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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.
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