2Overview of Talk Overview of proteomics as a concept Techniques discussion2D Gels and experimental design paradigmsProteomics mass spectrometryIdentificationQuantitation
3Proteomics is the study of the overall state of an organism’s temporal protein composition The biological state of the proteome is encoded inThe relative abundance of currently expressed proteins (and their isoform)Their localisation relative to cellular (or extracellular) structuresTheir interaction partner molecules and substratesTheir current post-translational modification stateTheir folded structures…
4A Different View on Life Genome Transcriptome Proteome … PhenotypeDifferent levels of biological complexityMore layers of regulation and controlIncreased heterogeneity of samples
5Why consider Proteomics? Orthogonal verification of gene activity.Observe biological state after more levels of regulation and control – closer to phenotypic outcome.Observe proteomes of extracellular locations – blood plasma/serum, urine etc.
6Proteomics Classical biochemistry Two-dimensional gels (2DGE) Mass spectrometryComputational analysis
7Methods in Proteomics Separation Identification Quantitation Gels ImmunochemistryChromatographyIdentificationMass spectrometryQuantitationAll of the above
8Identification vs Quantitation What’s there? How much of it is there?How sure are you about the ID?How sure are you about the abundance?Not there versus not detectable
92DGE Separate proteins by isoelectric point, then by mass Visualise with silver staining or coomassieUse CyDyes to label samples so they can be run together on the same gelAppl Microbiol Biotechnol October; 76(6): 1223–1243.
10Quantitation Experimental Paradigm - Labelling Label samples in such a way as to not affect subsequent processing but allow differentiation in final analysis. Examples:Fluorescent dyes (2DGE)SILAC amino acid labels (MS)Isobaric mass tags (MS/ MS)Process multiple samples simultaneously, differentiate only in final analysis on basis of label.Avoid some proportion of technical varianceBest to worst (for avoiding technical variance):Labelling in vivoLabelling protein mixtureLabelling peptide digestion mixture
11Aline Chrétien, Edouard Delaive, Marc Dieu, Catherine Demazy, Noëlle Ninane, Martine Raes, Olivier ToussaintUpregulation of annexin A2 in H2O2-induced premature senescence as evidenced by 2D-DIGE proteome analysisExperimental Gerontology, Volume 43, Issue 4, April 2008, Pages 353–359
12Quantitation Experimental Paradigm – Normalising to standard Combine each sample (labelled with one label) with a representative standard (labelled with another label).Perform analysisFor each protein in each run, normalise observed abundance in labelled sample to observed abundance in labelled standard.
14Mass SpectrometryMass Spectrometry is a technique for the detection and resolution of a sample of ions by their mass-to-charge ratio - represented by m/z where m is the mass in Daltons and z is the charge. ’
15Proteomic Mass Spectrometry Classical biochemistry techniques and 2DGE are, in general, ‘top-down proteomics’ – identify and quantify whole proteins.Most modern proteomic MS is ‘bottom-up’
16Shotgun/’bottom-up’ proteomics SeparationSDS-PAGEAntibody-based approachesLNDLEEALQQACEDLARN KLNDLEEALQQAKDigestionSeparationSCXHigh pH RP LCLow pH RP LCAnalysisMS-MS/Tandem MSLNDLEEALQQAKEDLARNKLNDLEEALQQAKNVQDAIADAEQRSKEEAEALYHSKSLVGLGGTKTAAENDFVTLKTAAENDFVTLKKTSQNSELNNMQDLVEDYKTSQNSELNNMQDLVEDYKKVDLLNQEIEFLKYEELQVTVGRYLDGLTAERADLEMQIESLTEELAYLKADLEMQIESLTEELAYLKKAETECQNTEYQQLLDIKPeptide IDs+ QuantitationIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIIPI:IPIObserved Proteins+ QuantitationProteinsPeptides
17Tandem Mass Spectrometry Intensitym/zMass Analyser + DetectorMass SpectrumSampleTandem Mass SpectrumMS/MS spectrumm/zIntensityMass Analyser + Detector
18Identification by MS/MS Mass Analyser + DetectorSearch fragment spectrum against a database of protein sequences. For each sequence, digest into peptides, generate an expected fragment ion spectrum, and match to observed spectrumm/zIntensity?m/zIntensityIITHPNFNGNTLDNDIMLIK
19Identification by MS/MS There are multiple commonly used MS/MS fragment spectra search engines, including:MascotSequestOMSSAX!TandemMS AmandaAndromedaProteinPilot
20A brief overview of Mass Spectrometric quantitation Please feel free to stop me and ask questions!
21Tandem Mass Spectrometry Intensitym/zMass Analyser + DetectorMass SpectrumSampleTandem Mass SpectrumMS/MS spectrumm/zIntensityMass Analyser + Detector
25Peptide Isotopomer Distribution This is all 1 peptideIntensitym/zThink of it as a frequency distribution based on a probability function.The relative intensity of each peak is the relative chance of a single peptide molecule having that m/z1/charge (z)