esc summer school cardiovascular sciences june...
TRANSCRIPT
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ESC Summer School Cardiovascular Sciences June 15, 2011
PROTEOMICS
Prof dr Anton J.G. Horrevoets
Molecular Cell Biology and Immunology
VU Medical Center Amsterdam
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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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MALDI-TOF MS
• Matrix-Assisted Laser Desorption Ionization Time Of Flight ms.
• sample in UV absorbingmatrix hit by UV laserpulse.
• peptideH+ desorbed, accelerated, enters field-free flight tube
• flight time = A.m1/2
• reflectron increases mass accuracy
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Sample 2D Page
Cut
Peptides Digest Isolate
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Peptide mass fingerprinting
100 e2d08_1 5 (0.965) Sb (10,20.00 ); Sm (SG, 2x6.00); Cm (1:9)
2042.009 TOF LD+
1.01e4
ion T9 2041.018
2699.112
int. 2043.000
2700.114
%
804.293
T2 814.471
T1 2698.110
T21 1729.894
2701.117
861.041
876.990
1066.069
T26-27 1363.661
T13 1274.593
1395.613
1462.738
1730.879
1806.886
1887.039
1888.029
2429.272
2044.030 T14-16 2428.278
2164.044
2165.063
2430.266
2691.145
2690.190
2702.119
2723.128
3174.552
3173.5643175.590
3176.578
3330.573
3685.717
T13-16 3683.695
3686.623
3687.688
0 m/z1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
m/z Ionisation efficiency !
814.489 861.105 1274.612 1363.655 1395.664 1729.908 1806.013 1806.869 1886.063 2041.015 2042.155 2163.046 2428.265 2690.126 2697.335 2698.127 2699.038 2719.405 2721.204 3172.451 3173.639 3682.893 3683.684
Protein dBase
Peptide dBase
compare
match
Example: HSP26_yeast
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SILS Mass spectrometry of biomacromolecules
2007 June 5th
Swammerdam Institute for Life Sciences
Electrospray LC-MSMS and sequence tag analysis
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What is a mass spectrometer?
A mass spectrometer isan instrument wherein
¾ Ions are generated ¾ Ions are separated as a
function of mass over charge ration m/z
¾ Ions are detected ¾ The mass over charge ratio
and the amount of ions are determined
Swammerdam Institute for Life Sciences
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Electrospray ionisation
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Electrospray ionisation
Swammerdam Institute for Life Sciences
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Quadrupole analyser
U VcosΩt
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ESI-TOF mass spectrum of Glu-fibrinopeptide B
536 537 538 5390
100
%
(M+2H)2+
(M+3H)3+ m/z
Swammerdam Institute for Life Sciences
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Protein sequencing by mass spectrometry
¾ electrospray ionisation of proteins and peptides
¾ MSMS principles and instrumentation to fragment gas phase ions
¾ amino acid sequence determination of peptides
Swammerdam Institute for Life Sciences
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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
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Collision-induced Dissociation
Collisions between ions with a certain kinetic energy and noble gas atoms or small molecules, such as N2 and O2.
Conversion of kinetic energy into internal energy results in dissociation of the mass selected ion.
N * F+
1
F+ 2
F+ 3
MH+ MH+
Swammerdam Institute for Life Sciences
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Assumed structures of ions formed by "backbone" fragmentation
X2 Y2 Z2
H2N CH R1
C O
NH CH R2
C O
NH CH R4
COOH + H
A2 B2 C2
A2 / a2 ions: H2N CH
R1
C O
N H
CH R2
B2 / b2 ions: H2N CH R1
C O
NH CH R2
C O
C2" / c2+2 ions: H2N CH R1
C O
NH CH R2
C NH3
O
C NH O
CH R3
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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:
X3 Y3 Z3 X2 Y2 Z2 X1 Y1 Z1
R1 O R2 O R3 O R4 H2N CH C NH CH C NH CH C NH CH COOH
A1 B1 C1 A2 B2 C2 A3 B3 C3
Biemann:
non-capital letters : a , b , c , x , y , zn n n n n n
instead of Y2"yn + 2
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MS-MS spectra of protonated peptides
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Format: 409,76-----T1A2G3-------528,13 mass1 internal sequence mass2
MSMS: classical sequence TAG
tag
Compare fragments
match 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600
M/z 0
100
%
990822 Sc band8Z 20%ACN sc08_20_1a MaxEnt 3 187 [Ev-30264,It20,En1] (0.036,187.07,0.169,1285.53,2,Cmp) 1: TOF MSMS 722.70ES+
(801.47) S S S Y (200.14) yMax 1444.78(M+H) +
820.48 y
722.42
136.08 Y
129.10
201.14 b 364.20
b1251.12
715.47 389.21 602.37 583.28
789.49
1081.59 y3994.56
y2907.52 y1
846.50 958.51
1045.57
1443.93 1244.64 y4
1208.62 1147.70 1400.88 1299.67
1445.32
1503.80
Internal sequence
dBase
Compare mass
shortlist
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Electrospray ionisation tandem mass spectrometry
Swammerdam Institute for Life Sciences
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Quantitative proteomics
How much do you see?
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A method for quantitative proteomics should …
• Be reproducible • Be reliable • Be sensitive • Cover many proteins • Have a large dynamic range
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ABSOLUTE vs relative
• Absolute : – Concentration – Copy number / cell
• Relative: – up – down regulation – comparison of states
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Methodology
• Gel based – Staining + Image analysis – Prederivatized proteins
• Without gels – Isotope free vs isotope labeling – Multi-dimensional LC – Affinity separations
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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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+
+
+
++
+++
1st Dimension: Iso Electric Focussing
pH3 pH10
+
--
---
+
-
+
+
+
-+ -
+
+
+ +
+
+---
-- -
-+
+ +
+
--
--
+
+ -+
-+
Time0
Time1
Time2
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2nd Dimension: SDS Page
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CyDyeCyDye DIGEDIGE fluorfluor StructureStructure
Fluor Fluor
linker + protein @ pH 8.5
O O
Lysine NHNHS ester active group N
H
Minimal labeling with Cy2,Cy3 or Cy5
Sensitivity: 125 pg/protein, linear response over 105 range. Compare: Coomassie >100 ng and Silver >10 ng.
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DIGE: Difference Gel Electrophoresis Protein extract 1 Protein extract 2Mix labeled extracts
Label with fluor 1 Label with fluor 2
Separate by 2-D PAGE
Excitation Image gel Excitation wavelength 1 wavelength 2
Analysis of difference Image analysis: overlay images Image analysis:
data quantitation ESC Summerschool 2011 Nice
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DeCyder procedure
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Gel based methods
Dynamic range Stain: 103 (?) 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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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)
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Protein Abundance Indices (PAI)
PAI 1.2
Nobsd PAI =
0.8 Nobsbl
0.4 Nobsd the number of observed peptides per protein 0.0
10 100 1000 10000Nobsbl the number of observable Protein concentration (fmol/μL)peptides per protein
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Isotope labels
Labels can be incorporated via:
1 Culture media 2 Derivatization of reactive groups on the
proteins 3 Proteolytic cleavage
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MS
Isotope ratio for quantification
Label introduction: techniques
Pool 1
Pool 2
Label 1
Label 2
derivatize
derivatize
digest
digest
MIX
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Isotope labels
• Extracted ion chromatograms (XIC) – Peak area of a selected m/z value
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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
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Examples of isotope post-hoc labels
H/D ICAT; D-labeled amino-acids (SILAC) Caution: D changes RP-HPLC retention
12C/13C ICAT the sequel 14N/15N Incorporated via growth media
Can be used to calculate # of nitrogens
16O/18O Incorporated by proteases Can be combined with other procedures
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Deuterium alters RP elution
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Isotope labels
Labels can be incorporated via:
1 Culture media 2 Derivatization of reactive groups on the
proteins 3 Proteolytic cleavage
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Spacer: X = H or D
Affinity Tag (Biotin)
ICAT structure
SH reactive D0:+C20H35N4SO5 / -H mass change = 442.225/cystein (mono) ; 442.5799 (av). D8:+C20H27D8N4SO5 / -H mass change = 450.2752/cystein (mono) ; 450.63 (av).
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ICAT procedure
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iTRAQ
Signal is concentrated in MS overview:
High sensitivity
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iTRAQ
Quantification on daughter ions in
MSMS:
Improved S/N ratio
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iTRAQ
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Isotope label methods
Dynamic range 103 : > 10 both up and down
Reproducibility Approximately 20% is attainable
Separating Thousands of PEPTIDES power (not equal to proteins) Bias Less biased than gels
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Correlation RNA/protein
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r = 0.61
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Or from the iTRAQ experiment
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Isotope labels
Labels can be incorporated via:
1 Culture media 2 Derivatization of reactive groups on the
proteins 3 Proteolytic cleavage
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18OInverse labeling with H2
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Trypsin exchanges two oxygens
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Equal and different amounts
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Examples of isotope labels
H/D ICAT; D-labeled amino-acids (SILAC) Caution: D changes RP-HPLC retention
12C/13C ICAT the sequel 14N/15N Incorporated via growth media
Can be used to calculate # of nitrogens
16O/18O Incorporated by proteases Can be combined with other procedures
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Literature
• Ong SE, Mann M. Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol. 2005 Oct;1(5):252-62.
• Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics. 2005 Sep;4(9):1265-72.
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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)
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Treatment of lysosomal storage diseases
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Discovery of chitotriosidase
Gaucher disease : search for markers
Patient plasma : 1000-fold increased 4-MU-chitotrioside hydrolysis
Unfortunately 10-20 % of people deficient for this enzyme
4-MU-chitotrioside I II III
100.000
10.000
1.000
100
10Chi
totr
ios i
dase
act
ivit y
( n
mo l
/ml/h
r)
0
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