PROTEOMICS IN VASCULAR BIOLOGY
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1 ESC Summerschool 2013 Nice ESC Summer School Cardiovascular Sciences June 18, 2013 PROTEOMICS IN VASCULAR BIOLOGY Prof dr Anton J.G. Horrevoets Molecular Cell Biology and Immunology VU Medical Center Amsterdam
2 1. Basics of proteomic mass spectrometry 2. Quantitative proteomics 3. New developments, Case studies
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9 1. Basics of proteomic mass spectrometry 2. Quantitative proteomics 3. New developments,. Case studies
10 The basics of MS
11 Identification S1 S2 S3 weak weak strong What is this protein band??? How would you solve this question? Coomassie SDS-PAGE
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13 What is a mass spectrometer? A mass spectrometer is an instrument wherein Ions are generated Ions are separated as a function of mass over charge ration m/z Ions are detected The mass over charge ratio and the amount of ions are determined Swammerdam Institute for Life Sciences
14 Many types of Mass spectrometers
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17 MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization Time Of Flight ms. sample in UV absorbing matrix hit by UV laser pulse. peptideh + desorbed, accelerated, enters field-free flight tube flight time = A.m 1/2 reflectron increases mass accuracy
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26 Peptide mass fingerprinting ion int. e2d08_1 5 (0.965) Sb (10,20.00 ); Sm (SG, 2x6.00); Cm (1:9) 100 % T T T T ESC Summerschool 2011 Nice T T T m/z Ionisation efficiency! T TOF LD+ 1.01e4 m/z compare match Protein dbase Peptide dbase Example: HSP26_yeast
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28 Protein sequencing by mass spectrometry electrospray ionisation of proteins and peptides MSMS principles and instrumentation to fragment gas phase ions amino acid sequence determination of peptides Swammerdam Institute for Life Sciences
29 Electrospray ionisation
30 Normal flow rate electrospray (top) vs a lower flow rate electrospray (bottom) that produces smaller droplets 100-fold ionization efficiency ESC Summerschool 2011 Nice Acc. Chem. Res., 37 (4), , 2004
31 Electrospray ionisation Swammerdam Institute for Life Sciences
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33 Collision-induced Dissociation Collisions between ions with a certain kinetic energy and noble gas atoms or small molecules, such as N 2 and O 2. Conversion of kinetic energy into internal energy results in dissociation of the mass selected ion. N MH + MH + * F + 1 F + 2 F + 3 Swammerdam Institute for Life Sciences
34 Peptide fragment ions Nomenclature: P. Roepstorff and J. Fohlman, Biomed. Mass Spectrom., 11 (1984) 601); K. Biemann, Biomed. Environ. Mass Spectrom., 16 (1988) 99. Roepstorff, Fohlman: X 3 Y 3 Z 3 X 2 Y 2 Z 2 X 1 Y 1 Z 1 R 1 H 2 N CH O C NH R 2 O CH C NH R 3 CH O C NH R 4 CH COOH A 1 B 1 C 1 A 2 B 2 C 2 A 3 B 3 C 3 Biemann: non-capital letters : a n, b n, c n, x n, y n, z n y n + 2 instead of Y 2 "
35 Assumed structures of ions formed by "backbone" fragmentation X 2 Y 2 Z 2 H 2 N R 1 CH O C NH R 2 O CH C NH R 3 CH O C NH R 4 CH COOH + H A 2 B 2 C 2 X 2 / x 2 ions: O C NH R 3 CH O C NH R 4 CH COOH Y 2 "/ y 2+2 ions: NH 3 R 3 CH O C R 4 NH CH COOH Z 2 / z 2 : R 3 CH O C NH R 4 CH COOH Other peptide fragment ions: a n+1, z n+1, d n, v n and w n ions (d n, v n and w n ions formed by "sidechain" fragmentation)
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37 Cleavage sites of trypsin in profilin AGWNAYIDNL MADGTCQDAA IVGYKDSPSV WAAVPGKTFV NITPAEVGVL VGKDRSSFYV NGLTLGGQKC SVIRDSLLQD GEFSMDLRTK STGGAPTFNV TVTKTDKTLV LLMGKEGVHG GLINKKCYEM ASHLRRSQY Swammerdam Institute for Life Sciences
38 MS-MS spectra of protonated peptides
39 Tandem mass spectrometry Recording of mass spectra of ions selected according to their m/z ratio. Swammerdam Institute for Life Sciences
40 MSMS: classical sequence TAG ESC Summerschool 2011 Nice dbase Format: 409, T 1 A 2 G ,13 mass1 internal sequence mass2 Internal sequence Compare mass Sc band 8 Z 20% ACN sc08_20_1a MaxEnt [Ev-30264,It20,En1] (0.036,187.07,0.169, ,2,Cmp) 100 (801.47) S S S Y (200.14) ymax (M+H) + 1: TOF MSMS ES+ tag shortlist y % Y b b M/z y y y y Compare fragments match
41 Quadrupole analyser U VcosWt Swammerdam Institute for Life Sciences ESC Summerschool 2011 Nice
42 Electrospray ionisation tandem mass spectrometry Swammerdam Institute for Life Sciences
43 1. Basics of proteomic mass spectrometry 2. Quantitative proteomics 3. New developments, Case studies
44 Quantitative proteomics How much do you see?
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46 Proteomic comparison - Are differences real (gel to gel and stainingvariations)? Possible solutions: pre purification, more gels, DIGE (Diff. gelelectrophoresis, cydyes : increases sensitivity) - Local stresses per gel differ... Solution: DIGE (again..)
47 A method for quantitative proteomics should Be reproducible Be reliable Be sensitive Cover many proteins Have a large dynamic range
48 ABSOLUTE vs relative Absolute : Concentration Copy number / cell Relative: up down regulation comparison of states
49 Methodology Gel based Staining + Image analysis Prederivatized proteins Without gels Isotope free vs isotope labeling Multi-dimensional LC Affinity separations
50 Gel based methods 1. Stain (CBB/Ag/Sypro) 2. Image analysis 3. Repeat for reproducibility Alternatively: Fluorescent derivatization prior to 2D-GE
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52 1 st Dimension: Iso Electric Focussing ph3 ph Time Time Time 2
53 2 nd Dimension: SDS Page
54 CyDye DIGE fluor Structure Fluor Fluor linker NHS ester active group + ph 8.5 O N H Lysine NH O Minimal labeling with Cy2,Cy3 or Cy5 Sensitivity: 125 pg/protein, linear response over 10 5 range. Compare: Coomassie >100 ng and Silver >10 ng. ESC Summerschool 2011 Nice
55 Protein extract 1 Label with fluor 1 DIGE: Difference Gel Electrophoresis Mix labeled extracts Protein extract 2 Label with fluor 2 Separate by 2-D PAGE Excitation wavelength 1 Image gel Excitation wavelength 2 ESC Summerschool 2011 Nice Image analysis: overlay images Analysis of difference Image analysis: data quantitation
56 DeCyder procedure
57 Sample 2D Page Cut Peptides Digest Isolate
58 Gel based methods Dynamic range Stain: 10 3 (?) Fluor:??? Reproducibility Stains: worse than 2 Fluor: up to 1.3 (ideal) Separating power Thousands of SPOTS (not equal to proteins) Bias Soluble, abundant
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61 Isotpe labelling
62 Isotope labels or isotope free Mass spectrometry is not quantitative Ionisation propensity Co-suppression Co-enhancing Use of (stable) isotopes serves as an internal standard (chemically identical)
63 Isotope labels Labels can be incorporated via: 1 Culture media 2 Proteolytic cleavage 3 Derivatization of reactive groups on the proteins
64 Label introduction: techniques Label 1 Pool 1 derivatize digest MIX Pool 2 derivatize digest MS Label 2 Isotope ratio for quantification
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66 Examples of isotope labels H/D ICAT; D-labeled amino-acids (SILAC) Caution: D changes RP-HPLC retention 12 C/ 13 C ICAT the sequel 14 N/ 15 N Incorporated via growth media Can be used to calculate # of nitrogens 16 O/ 18 O Incorporated by proteases Can be combined with other procedures
67 Isotope labels Extracted ion chromatograms (XIC) Peak area of a selected m/z value
68 14 N/ 15 N : LCMS Mass shift: number of nitrogen's in the peptide
69 Yeast cell wall proteins labeled with 15 N ESC Summerschool 2011 Nice
70 Isotope label methods Dynamic range 10 3 : > 10 both up and down Reproducibility Separating power Bias Approximately 20% is attainable Thousands of PEPTIDES (not equal to proteins) Less biased than gels
71 When to mix in the label? Culture media: -early in the procedure -identical treatment throughout the preparation process -keeping standards standard may be difficult Chemical probes/digestions -irreproducibility's: check by inverse labeling
72 ICAT structure Spacer: X = H or D SH reactive Affinity Tag (Biotin) D0:+C 20 H 35 N 4 SO 5 / -H mass change = /cystein (mono) ; (av). D8:+C 20 H 27 D 8 N 4 SO 5 / -H mass change = /cystein (mono) ; (av).
73 ICAT procedure
74 itraq Signal is concentrated in MS overview: High sensitivity
75 itraq Quantification on daughter ions in MSMS: Improved S/N ratio
76 itraq
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78 1. Basics of proteomic mass spectrometry 2. Quantitative proteomics 3. New developments, Case studies
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83 Gaucher Disease Lysosomal storage disorder Deficiency in lysosomal glucocerebrosidase Substrate : glucosylceramide Flux of glycosphingolipids is largest in monocyte-macrophage system These cells become major storage sites (Gaucher cells >> mainly found in liver, spleen and bone marrow)
84 Treatment of lysosomal storage diseases
85 Discovery of chitotriosidase Gaucher disease : search for markers Patient plasma : 1000-fold increased 4-MU-chitotrioside hydrolysis Unfortunately % of people deficient for this enzyme MU-chitotrioside I II III 0
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90 Post-translational modifications
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95 Kinome analysis: Symbol Phosphosite Bayes.p Fold Symbol Phosphosite Bayes.p Fold FAK Y PKCg T FAK S Abl Y CDK1/2 T14+Y RSK1/2 S363/S Hsp27 S Src Pan-specific FKHRL1 T HO2 Pan-specific Hsp27 S ATF2 T51+T CDK7 Pan-specific p38a Pan-specific HspBP1 Pan-specific PKCb2 T c-jun S STAT5A Y Crystallin ab S S6Ka T IRAK4 Pan-specific Bad S PKCq S EGFR Pan-specific Ksr1 Pan-specific PKCg Pan-specific Rb S FAK Y PP6C Pan-specific Erk4 Pan-specific Tau S Kit Y EGFR Y ERK5 T218+Y Boon, Blood 2010
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98 Glycosylation of IgG changes during disease: biomarkers
99 Glycosylation structure determination using MS-MS
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102 EPC = monocyte with ingested platelets!
103 EPC = monocyte with ingested platelets!
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106 Proteomics
107 metabolomics
108 Nothing changed at metbolite level!!: Homeostasis Combined tri-omics: increased sensitivity
109 transcriptomics
110 Combined tri-omics: increased sensitivity
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114 Carotid OPN predicts all vascular events!
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