Strategies for Quantitative Proteomics. Atelier "Protéomique Quantitative" La Grande Motte, France - June 26, 2007

Transcription

1 Strategies for Quantitative Proteomics Atelier "Protéomique Quantitative", France - June 26, 2007 Bruno Domon, Ph.D. Institut of Molecular Systems Biology ETH Zurich Zürich, Switzerland OUTLINE Introduction Proteomic strategies Shot-gun vs. targeted approaches Sample preparation Reduction of sample complexity Labeling techniques Mass spectrometry platforms High-performance LC/MS MRM technique Multiplexed analyses Conclusion 2 1

2 Challenges in Biomarker Analysis Complexity of plasma proteome > 50,000 proteins (> several mio peptides ~ shotgun approach) Exceed peak capacity of any LC/MS system Reduction of complexity: Target a sub-proteome Fractionation Wide dynamic range in plasma Protein concentrations span over ten orders of magnitude Typical dynamic range of most LC/MS systems 3-4 logs (gap!) High performance analytical platform with increased resolution, sensitivity and dynamic range. 4 Limitations of Shotgun Approach Proteins observed in plasma Sample complexity exceeding LC/MS peak capacity Under-sampling (random MS/MS) Bias towards abundant components (lack of depth) Identification and quantification 7 2

3 Shotgun LC/MS/MS Conventional shotgun strategy: Identification and quantification are coupled!! Only most abundant peptides are identified and quantified 11 Directed MS/MS Experiment

4 Alignment of LC/MS Profiles Data Analysis Feature Detection Quantification Alignment Clustering Features LC/MS Maps Elution time (x) m/z (y) Intensities (grey scale) Peptide Array Differentials Clustering Samples 16 Sample Preparation (N-glycosites) Capture Wash Trypsin digestion Wash PNGaseF Digestion N-Glycosites Asp ( Asn) Solid phase capture of glycopeptides Reduction of sample complexity (20 fold) 20 4

5 Reduction of Complexity # Peptides N-linked glycopeptides Tryptic peptides N-Glycosites Tryptic peptides Human Protein DB Fully tryptic peptides NXS/T peptides MMass 5800 Glycopeptide capture (N-linked): ~ 20 fold reduction of complexity 21 Off-Gel Electrophoresis Fractionation 10 9 pi Off-gel electrophoresis principle OGE fraction Calculated pi of peptides identified 22 5

6 II. Target Proteotypic Peptides Samples Sample Prep LC-MS/MS: MRM List of MRM ions > 1000 Pep Atlas Seq,, Prot,RT,MM, z STRATEGY Use MRM for hypothesis-driven discovery Generate list of proteotypic peptides Predict transitions & elution times Perform wide MRM screen (~1000 ions) Quantification Confirmation of ID Proteins Transcripts Literature 27 Peptide to Protein Inference Proteotypic peptide Unique to one protein Observable by LC/MS Ambiguous peptide 28 6

7 Multiple Reaction Monitoring (MRM) High selectivity ~ two levels of mass selection (increased S/N) High sensitivity because of high duty cycle (Q1 and Q3 are static) Increased dynamic range Only known peptides are detected Source MS-1 CID MS-2 Fixed Set precursor m/z Peptide (M) Fixed Set fragment m/z Fragment (m) time 29 MRM Transitions LDVDQALDR Rel. Int m/z P y a y b Select high intensity transitions Use two transitions per peptides Add internal standard (labeled peptides) P y a P* y a * 32 7

8 Quantification (IS added to Serum) 4.0E E+07 2.E+05 1.E+05 5.E+04 = 35 amol 526/ 609: LDVDQALDR 609/ 821: TJFPDLTDVR 444/ 502: DGTLVAFR 2.0E fmol injected 1.0E+07 N-glycosite fraction Dynamic range > 4.5 log LOQ < 50 amol: < 0.1 ng/ml fmol injected 33 Biomarker Discovery Strategy Differentiation healthy from disease serum proteomes Label free strategy Isotope labeling strategy N-Glycosite capture N x LC-MS Anal. Pool Capture Light 296: 113 Labeling Heavy 304: 121 Data Analysis Combine LC/MS/MS 1x LC/MS Analysis 45 8

9 Stable Isotope Labeling 56 itraq Reagent 57 9

10 itraq Tandem Mass Tags: Multiplexing Reporter Balance Reactive group Charged Neutral Amine specific Methyl-piperazine H 2 N-Peptide NHS ester Mix 4 samples & MS/MS analysis MS/MS spectrum m/z MReporter + MBalance = Constant Labeling of amino groups Multiplexed analyses (four) All precursors have same mass No increase in sample complexity Signal amplification Quantification in MS/MS mode 58 Conclusion Targeted mass spectrometry strategies Increase depth of the proteome MRM technique: high sensitivity and selectivity Shift paradigm for discovery using hypothesis-driven screen Combination of techniques "to dig deeper" Effective enrichment and fractionation methods Targeting N-glycosite subproteome Fractionation through electro-focusing Robust and high performance MS techniques High mass accuracy Reproducible chromatography. Robust platforms Identification and quantification of biomarkers in serum or plasma