NPC Progress Meeting 2012 - Netherlands Proteomics Centre

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Figure 2 | Targeted proteomics. (A) Definition of time windows in scheduledSRM. Each peptide elutes at a specific retention time and therefore eachpeptide is only measured across a narrow time window. (B) Relative quantificationof the targeted peptides. An example of how the relative concentrationof a peptide that decreases over time can be quantified using the ratioto a reference mixture added at equal concentrations to all samplesmultiple fragments to further increase specificity, and multiplepeptides per protein are followed to increase the confidenceon the protein level.Assay development Clear advantages of targeted proteomicsare a decreased need to reduce sample complexity and theincreased dynamic range over which proteins can be detectedand quantified. However, the development of assays requiressubstantial effort compared to the discovery-based proteomicsapproaches. For the development of assays three steps needto be performed, namely protein target selection, selection ofsuitable peptides and selection of the peptide fragments that18 | NPC Highlights 15 | Rainer Bischoffwill be monitored. Selection of the protein targets is crucial,because no information will be gathered outside the selectedtargets (in contrast to discovery-based proteomics).Multiple peptides (3-5) are preferably used for each targetprotein. These peptides are selected taking into account thatthe peptides have to be unique for the protein target (proteotypic)and that they are suitable for (reversed phase) LC separationand MS detection (MS signal response, size, charge). Foreach peptide, the best peptide fragments (3-5) are then selectedfor identification and quantification. The best peptideand peptide fragment combinations within the workflow canbe determined using synthetic peptides, but information canalso be obtained from (public) MS/MS data repositories. Publiclibraries like the NIST library (http://peptide.nist.gov) andPeptideAtlas (www.peptideatlas.org) contain MS/MS spectrafor several organisms.Separation of complex peptide mixtures on the LC columnincreases the number of peptides that can effectively bemeasured in a single run. Each peptide elutes at a specificretention time (based on its physico-chemical properties, thetype of column and type of gradient) and therefore each peptideis only measured across a narrow time window (see Figure2A). In modern triple quadrupole mass spectrometers, dutycycles are sufficiently fast to monitor 150 peptide fragmentsconcurrently.Relative quantification Stable isotope labelled syntheticpeptides (heavy peptides) have to be spiked (added to thesample) for quantification of the peptide concentrations incomplex samples. The heavy peptides will have the sameretention times and ionisation properties as the target (light)peptides and can therefore be used to correct for variationswithin the method and for matrix effects. However, synthesisof individual peptides will become costly for large numbers oftargets.Alternatively, relative quantification can be used. Relativequantification is well suited for studying replicative ageing,since we want to compare the mother cells after various numbersof replication cycles. Figure 2B shows an example of howthe relative concentration of a peptide that decreases overtime can be quantified using the ratio to a reference mixtureadded at equal concentrations to all samples. As referenceyeast cells were grown with a 15 N nitrogen source and thesecultures are used for spiking. Careful consideration is requiredto create an appropriate reference sample which contains allproteins of interest in 15 N-labelled form. In our case we haveharvested yeast cultures at different growth phases (exponentialphase, cells after diauxic shift and cells that are in thestationary phase) to create a global internal standard.Cellular processes Targeted proteomics is especially suitableto comprehensively study alterations in all proteins involved inspecific pathways which can show a large range of concentrations[4]. In a collaborative project within the Systems BiologyCentre for Metabolism and Ageing (SBC-EMA) in Groningen,we will combine the obtained protein levels with measurementsof metabolite levels as well as the determination of theage-associated phenotype at the single cell level. Currently,assays have been developed for about 350 proteins, whichcan be detected with 3-5 peptides each. The proteins werechosen to cover all biological processes as described by theirGene Ontology function and included proteins that have beendescribed in the literature as being affected during ageing.The application of targeted proteomics to the study of cellularprocesses is illustrated in Figure 3. Here, 54 proteins thatare involved in glycolysis and gluconeogenesis were targetedin yeast cultures grown to mid-exponential phase, after thediauxic shift, and at the stationary phase. Proteins werequantified relative to a digested reference standard createdby combining cultures grown with a 15 N nitrogen source andharvested at these three phases. The concentration of severalproteins shows a clear dependence on the growth phase.
Even more interesting is the different behaviour of variousisoenzymes, as is observed, for example, for the phosphoglucomutasesPGM1 (YKL127W) and PGM2 (YMR105C) in the upperpart of Figure 3.From yeast to mice The methods described above werespecifically developed for yeast proteins. However, similar approachescan be adopted to create assays for other organisms,as long as sequence information (and preferably MS/MS information)is present or can be obtained experimentally. We arecurrently developing assays to measure the effect of ageing inthe mouse proteome.References1 Partridge, L. (2011) Some highlights of research on ageingwith invertebrates 2010. Ageing Cell 10 (1), 5-9.2 Domon, B. and Aebersold, R. (2010) Options and considerationswhen selecting a quantitative proteomics strategy.Nature Biotechnology 28 (7), 710-721.3 Lange, V. et al. (2008) Selected reaction monitoring forquantitative proteomics: a tutorial. Molecular SystemsBiology 4 (222), 1-14.4 Picotti, P. et al. (2009) Full dynamic range proteome analysisof S. cerevisiae by targeted proteomics. Cell 138 (4),795-806.| 19Figure 3 | Metabolic pathways in yeast measured with targeted proteomics. Relative proteinconcentrations in yeast cultures at various growth phases for proteins involved in the glycolysis andgluconeogenesis pathway (according to the KEGG database (www.kegg.jp)). The three coloured blocksindicate the ratios of the ( 14 N) mid-exponential growth phase (14M), the diauxic shift (14D) and thestationary phase (14S) versus the ( 15 N) standard culture. Ratio colours range from dark red (2) indicatingthat the protein is abundant.Research teamUniversity of Groningen: Karin Wolters, postdoc at theSystems Biology Centre; Hjalmar Permentier, head of the MassSpectrometry Centre; and Rainer Bischoff, chair Analysis ofBiological Macromolecules.ContactProf. Dr. Rainer BischoffGroningen Research Institute of PharmacyAnalytical BiochemistryAntonius Deusinglaan 19713 AV Groningen, the NetherlandsT +31 50 363 3338r.p.h.bischoff@rug.nlsummaryTargeted proteomics has great potential for studying thechange of protein levels in biological processes more robustlythan discovery-based proteomics. The presented example ofprotein abundances in the yeast glycolysis pathway in variousgrowth phases illustrates the wealth of information that canbe obtained. These data will be combined with metabolomicsdata and single cell analyses to create a more comprehensiveimage of replicative ageing in yeast in a systems biologyapproach. Our study of the changes in protein levels duringreplicative ageing in the mother cells is part of a concertedeffort to study the molecular mechanisms of replicativeageing at both the single cell and the population level at theSystems Biology Centre for Metabolism and Ageing (SBC-EMA,one of three national systems biology centres funded bythe Netherlands Organisation for Scientific Research). Dataon protein and metabolite levels are obtained by MS-basedtargeted proteomics and metabolomics. This approach will beextended to study the effect of environmental conditions suchas dietary changes and other stress conditions.
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