1
Differential Protein Expression Analysis
Robert Cole, Ph.D.
371 Broadway Research [email protected]
Mass Spectrometry/Proteomics Facilityat Johns Hopkins School of Medicine
The Core Facility
Gel Electrophoresis Protein Digestion + Processing Mass Spectrometry
DIGE WorkshopIn collaboration with
GE HealthCare
Hands-on Training in 2D and DIGE SDS-PAGEParticipants analyze real samplesOffered quarterly
2
Walkup use of MALDI-TOF
MALDI-TOF Training:Learn to operate MALDI-TOFOffered Thursdays by appointment
LCQ Deca XP
Plus
Services• Consultation • 2D Gel Electrophoresis • Mass Spectrometry Analysis (PMF, Tandem)• Protein Identification and Modifications
(isolated proteins, gel spots, complex mixtures) • Protein Quantification
(DIGE, iTRAQ or 18O-labeling analysis) • MALDI-TOF Training• LCQ Workshop • DIGE Workshop • iTRAQ Workshop
Why Protein Expression Analysis?
Quantify protein expression between normal vs disease samples
Quantify protein expression between wild type and mutant cell lines
Quantify proteins present in protein complexes
Determine protein subcellular localization
Follow protein trafficking
Map metabolic pathways
…many, many others
3
Differential Protein Expression Analysis
Topics
Protein identification by mass spectrometry
Differential protein expression techniques
Sample preparation
Protein Identification Workflow 1
Databasesearch
Separation IdentificationDigestion Analysis
Coomassie BlueSilver StainFluorescent Dye
TrypsinPeptide Masses
Peptide Mass Fingerprint(PMF)
uses just peptide masses
1D Gel
2D Gel
HPLC
1990.2
Mass to charge ratio (m/z)
0
50
100
% In
tens
ity
500 1200 1900 2600 3300 4000
831.5985.6
1141.7
1398.91555.0
1875.2
2048.1 2414.4
2558.4
2870.6
3319.7
1972.1
1578.91381.8
Peptide Masses Recovered from an Gel Band
4
Database Searching with Peptide Masses depends on
Mass Accuracy and Protein Coverage
Peptide Mass ppM # Match Proteins
1975.95 100 20010 44
1526.88 100 19710 19
1055.54 100 51410 87
100 ppm = 0.10 Da per 1000 Da10 ppm = 0.01 Da per 1000 Da
Protein Identification Workflow 2
Databasesearch
Separation IdentificationDigestion Analysis
2 - Peptide FragmentsCoomassie BlueSilver StainFluorescent Dye
Trypsin1- Peptide Masses
EACDPLR
Tandem MSor
MS/MS
uses peptide mass+ sequence
1D Gel
2D Gel
HPLC
Tandem MS (or MS/MS)
Uses Peptide Mass and Sequence Tag
Mass Accuracy <100 ppm
One Peptide (at least two preferred)
>3 amino acids masses in sequence
Complex protein samples
Protein modifications identified and mapped to an amino acid
PMF
Uses Peptide Masses
Mass Accuracy <100 ppm
Protein Coverage >30%
Low Sample Complexity
No more than three proteins
Protein modifications suggestedby a change in peptide mass
PMF vs. Tandem MS
5
How much protein do you need?
-100 kD
- 50 kD
- 25 kD
-220 kD
10 20 30 40 50 100 ng
Silver stained gel
100 fmol per protein – in gel band or spot10 fmol per protein – in solution
Search Engines for Protein Identification from MS Data
Summary of ProgramsProteome Software www.proteomesoftware.com/ExPASy expasy.proteome.org.au
Free ProgramsProteinProspector prospector.ucsf.eduXProteo xproteo.com:2698Prowl prowl.rockefeller.eduMascot www.matrixscience.com (Free up to 300 ions)
Open Source ProgramsOMSSA pubchem.ncbi.nlm.nih.gov/omssa/X! Tandem www.thegpm.org/
Commerical ProgramsMascot www.matrixscience.com Sequest fields.scripps.edu/sequest/Spectrum Mill www.chem.agilent.com/Proteolynx www.waters.com/WatersDivision/
Topics
Protein identification by mass spectrometry
Differential protein expression techniquesDifferential protein expression techniques
Sample preparation
6
Differential Protein ExpressionProtein Pre-Fractionation (sample complexity)
(low abundance proteins)Protein/Peptide Labeling
Fluorescent tag (Cy dyes)Stable isotope (13C, 15N, 18O)Mass tags (Signature mass)
Protein/Peptide SeparationGel Electrophoresis (MW, pI)Chromatography (MW, charge, affinity)Both
Quantification (comparison of label)
Protein Identification (by mass spectrometry)
Sample Preparation Must Be Reproducible!
Replicates 1 2 3 1 2 3
Poor! Good!
Gel Based Techniques
7
2
1D Gel AnalysisGel slice number Name of protein
22 Polymeric immunoglobulin receptor
22 Transferrin22 Vanin 1
21 1B-glycoprotein
21 Complement component 5
21 hGC-1 (human G-CSF-stimulated clone-1)
21 IgG Fc-binding protein21 Mac-2-binding protein
18 -1-antichymotrypsin18 Albumin
17 Amylase, pancreatic, -2A17 Amylase, salivary, -1A17 Catalase
Sample
Slice
Kristiansen et al. Molec Cell Proteomics 3:715–728, 2004.
1
1D Gel Analysis
Strengths:
• Simple
• Cheap
• Membrane proteins
• High MW proteins (>100 kDa)
Limitations:
• Time consuming
• Limited resolution
• Low MW Proteins (<7kDa)
• Not Quantitative
Principle of 2-D Gel Electrophoresis
First dimension:according to the isoelectric point (isoelectric focusing)
Second dimension:separation according to molecular weight (SDS gel electrophoresis)
8
Bacterial Strain #1 Bacterial Strain #2
2D Gel Analysis
Major problem with reproducibility and matching spots
pIMW
Gel Based Techniques with
Fluorescent Tags
Difference Gel Electrophoresis (DIGE)Disease sampleLabel with fluor 2
Control SampleLabel with fluor 1
Separate proteins on one 2-D gel
Image gelExcitation wavelength 1
Excitation wavelength 2
View differences
Mix samples
Protein comparison in a single gel!
9
Cy3 Labeled Glial-line 1 Proteins3.0 10.0IEF SD
S-PAG
E
250-150-
100-
75-
50-
37-
25-
20-
Cy5 Labeled Glial-line 2 Proteins3.0 10.0IEF SD
S-PAG
E
250-150-
100-
75-
50-
37-
25-
20-
Overlay of Glial-lines 1 and 2 Gel Images
3.0 10.0IEF SDS-PA
GE
250-150-
100-
75-
50-
37-
25-
20-
10
Three Fluorescent Cy Dyes
Cy2, Cy3, Cy5
ε-amino group of lysine
Matched for charge (carries +1 charge)
Matched for MW (~450 Da)
Only 3-5% of proteins are labeled (minimal labeling)
Cy2
Cy5
Cy3
Disease sampleLabel with Cy5
Control SampleLabel with Cy3
Separate proteins on one 2-D gel
Excitation wavelength 1
Excitation wavelength 2
View differences
Mix samples
DIGE Analysis Pooled Samples labeled with Cy2(on all gels to compare one gel to another)
Differential Protein Expression Between Two Glial Cell Lines
250-150-
100-
75-
50-
37-
25-
20-
3.0 10.0IEF SDS-PA
GE
11
Third Cy Dye (Cy2) Labeling as an Internal Standard
All possible protein spots overlaid on every gel.
Simplifies gel to gel matching.
Each spot has it’s own internal standard spot for normalizing across gels.
Reduces experimental variations.
Accounts for differences in sample load.
Differential Protein Expression Between 1st and 3rd Trimester Sera
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
12
DIGE Analysis of Ecoli proteins spiked with four proteins
Albumin0.5 X Conalbumin
100 X
Phosphorylase b0.5 X
Trypsin Inhibitor4 X
250-150-
100-
75-
50-
37-
25-
20-
4 7IEF SDS-PA
GE
Relative Abundances of Four Proteins in Ecoli Lysates are within 10% of Predicted Values
0.000144%3.844Trypsin inhibitor
1.3E-108%0.460.5Phosphorylase B
1.6E-1410%89.67100Conalbumin
2.7E-063%0.510.5Albumin
t-testErrorObserved Ratio
Expected Ratio
Spiked Proteins
Strengths
• Superb resolving power
• Quantitative comparisons of ~2000 proteins on one gel
• Simplified comparison between gels
• Highly reproducible
• Detect post-translationalmodifications
Limitations
• <7,000 and >130,000 Da
• Strongly hydrophobic Highly basic proteins
• Difficult to automate
Strengths/Limitations of 2D-DIGE Gels
13
Mass Spectrometry Based Techniques
Multi-dimensional Chromatography
Link et al. Nat. Biotech 17:676-682, 1999
Denatured proteins
Digest
Peptide mixLoad
Peptide ID by Tandem MS
2D chromatographyseparations of
peptides
Col1
Col2
Peptides Identified
Col2only Col 1 and Col2
219
115
(Shotgun or MudPit)
Summary of Multi-Dimensional Chromatography
Strengths:• Reduces sample complexity
• Low abundance proteins
• Membrane proteins
• All MW Proteins
• Flexible (different column combinations)
• Can automate
Limitations:• Complex setup
• No faster than 2D gels
• Computer intensive
• Not Quantitative
• Reproducibility
14
Mass Spectrometry Based Techniques
with Stable Isotopes or
MassTags
Three Basic Methods
Enzymatic: 18O-Labeling with Trypsin
Chemical: ICAT (Isotopic Coded Affinity Tags)iTRAQ (Isobaric Tags for Relative and
Absolute Quantitation)
Metabolic: SILAC (Stable Isotope Labeling of Amino acids in Cell culture)
18O-labeling Protocol
Disease sampleControl sample
Mix labeled peptides
Analyze by mass spectrometry
Trypsin Trypsin + 18O-water
C-terminal C-terminal(adds 4 Da)
15
+TOF MS: Experiment 1, 25.186 to 25.314 mi...a=3.56446231548684480e-004, t0=-4.886713...
Max. 2290.5 counts.
581.0 582.0 583.0 584.0 585.0 586.0 587.0 588.0m/z, amu
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
Inte
nsity
, cou
nts
584.32
584.82
582.32
585.33
Relative Quantification from Full Mass Spectrum
(Ratio of 18O to 16O doubly charged peptides should be 4)
Quantifying Peptides by 18O-Labeling
Intensity
m/z (mass divided by charge)
Strengths/Limitations of 18O-Labeling
Strengths:
• No extra steps
• Relative quantification using multiple peptides from same protein
• Post-translationalmodification information
• 18O labeling kit (Prolytica)
Limitations:
• No reduction in sample complexity
• Only 4 amu mass increase
• 18O exchange with water
• Quantification error ~20%
• No good software yet
Isotope–Coded Affinity Tags(ICAT)
SH
cys
Binds to Cysteines
Biotin Linker
heavy or
light isotope
S
HN NHO
NH O O
NH
IO OX
X
X
X
X
X
X
X
Gygi et al .Nature Biotech 17: 994-999, 1999
Reduce sample complexity.
Analyze only cysteine-containing peptides.
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Isotope-Coded Affinity Tag (ICAT)(cysteine-containing peptides)
550550 560560 570570 580580
mass
Light
Heavy
Hansen et al. Molec Cell Proteomics 2:299–314, 2003Gygi et al .Nature Biotech 17: 994-999, 1999
Strengths/Limitations of ICAT
Strengths:• Overcomes many 2D gel
limitations
• Relative quantification
• Reduces complexity of protein digest (only cysteine containing peptides)
• Low abundance proteins
• Compatible with gels
Limitations:• ~20% of proteins
lack cysteines
• Protein identifications often based on one peptide
• No post-translationalmodification information
• Quantification error ~20%
• Multi-step process, peptide losses
iTRAQ Tags(Isobaric Tag for Relative and Absolute
Quantitation)
Peptide Reactive Group
(binds to amines)
Reporter Balance PRG
ReporterMass114115116117
BalanceMass
31302928
Isobaric Tag(Total mass = 145)
Four possible tags
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The Structure
Three Disease Protein SamplesControl Protein
Sample
Mix Tag 114, 115, 116, and 117 Labeled Peptides
Digest
Label Peptides with Tag 114
Digest
Label Peptides with Tag 115
Digest
Label Peptides with Tag 117
Digest
Label Peptides with Tag 116
Analyze SCX Fractions by LC-MS/MS
Fractionate Peptide Mixture on SCX Column
iTRAQ Workflow 1
iTRAQ_KL_100ug
-150
-50
50
150
250
350
450
0 10 20 30 40 50 60 70
Time (min)
Abs
orba
nce
(mA
U)
How many
fractions to
collect?
Strong Cation Exchange (SCX) Chromatography
Of iTRAQ Labeled Mitochandria Peptides
Depends on
sample complexity
speed of mass spectrometer
18
Base Peak Chrom. of +TOF MS: Experiment 1, from 050822_VanEyk_KL_fraction_... Max. 4234.0 cps.
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95Time, min
0
500
1000
1500
2000
2500
3000
3500
40004234
Inte
nsity
, cps
578.95528.33
473.60
581.35
411.74549.99410.75
523.75
415.20404.74 536.11
536.11
Peptide Elution Profile: SCX Fraction 3 of 6
Base Peak Chrom. of +TOF MS: Experiment 1, from 050926_VanEykJ_KL_K10.wif... Max. 2556.0 cps.
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
24002556
Inte
nsity
, cps
467.62
431.60 475.63
639.37
400.27
498.55
431.93581.10
951.87454.77
Peptide Elution Profile: SCX Fraction 10 of 24
40 min
+TOF MS: Experiment 1, 40.039 to 40.775 min ...a=3.56446231548684480e-004, t0=-4.8867138...
Max. 257.6 counts.
400 450 500 550 600 650 700m/z, amu
0
20
40
60
80
100
120
140
160
180
200
220
240
258
Inte
nsity
, cou
nts
427.29 643.42
482.82465.07
498.83421.06
Mass Spectrum of iTRAQ Labeled Peptides Eluting at 40 min
19
+TOF Product (643....a=3.564462315486...
Max. 524.0 counts.
200 400 600 800 1000m/z, amu
0
100
200
300
400
500
Inte
nsity
, cou
nts
371.27
420.80 900.51357.27 464.32
Fragmentation Spectrum of 643 Ion
+TOF Product (643....a=3.564462315486...
Max. 524.0 counts.
114.0 115.0 116.0 117.0m/z, amu
0
20
40
60
80
100
120In
tens
ity, c
ount
s
117.12
116.12
115.11
114.11
Expanded Reporter RegionRatios to 114
Should be 2, 5 and 10
Validation of Relative Protein Ratios using iTRAQ
10 ug5 ug2 ug1 ugBSA
10 ug5 ug2 ug1 ugB-galactosidase
10 ug5 ug2 ug1 ugA-lactalbumin
10 ug5 ug2 ug1 ugB-lactoglobulin
10 ug5 ug2 ug1 ugLysozyme
10 ug5 ug2 ug1 ugApotransferrin
1:5
Sample 3116
1:101:21:1Expect Ratio to Sample 1
Sample 4117
Sample 2115
Sample 1114
ProteinMix
iTRAQ analysis shows relative protein ratios
not absolute protein ratios
4.63.41.8Apotransferrin
6.04.11.9Lysozyme
8.55.02.5B-lactoglobulin
5.93.61.6A-lactalbumin
6.13.61.8B-galactosidase
6.43.71.8BSA
Ratio 117:114(10 expected)
Ratio 116:114(5 expected)
Ratio 115:114(2 expected)
ProteinMix
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Two treated samples
Mix Tag 114, 115, 116, and 117 Labeled Peptides
Digest
Label Peptides with Tag 114
Digest
Label Peptides with Tag 115
Digest
Label Peptides with Tag 117
Digest
Label Peptides with Tag 116
Analyze SCX Fractions by LC-MS/MS
Fractionate Peptide Mixture on SCX Column
iTRAQ Workflow 2Two controls
Using iTRAQ to Identify and Quantify Proteins Expression of Mouse MyocaridalMitocohondria Proteins After Morphine
Treatment
Collaboration with Leslie Kane and Jenny Van Eyk at Johns Hopkins University
2) Mice treated 5 days with placebo or morphine
714621679731Total Protien (ug)
for iTRAQ
M2M1P2P1Sample
P1 P2 M1 M2M
1) Mouse Groups: Placebo 1 and 2 (P1,P2) Morphine treated 1 and 2 (M1, M2)
BCA protein quantification
5µg per lane
3) Extract Mitochondria
4) Remove aliquots to assay:
A-Total protein amount B-Reproducibility of sample preparation by gel electrophoresis
5) iTRAQ Labeling: P1 – 114P2 – 115M1 – 116M2 - 117
6) SCX Fractionation
7) LC-MS/MS Analysis
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Protein Ratios Based on Multiple PeptidesN Accession Protein Name Ratio 115:114 Ratio 116:114 Ratio 117:114
1 gi|20455479 ATP synthase beta chain, mitochondrial precursor 0.9085 1.0834 1.10462 gi|416677 ATP synthase alpha chain, mitochondrial precursor 0.9314 0.925 0.92083 gi|51338804 NAD(P) transhydrogenase, mitochondrial precursor (Pyr 0.9905 0.9487 0.91424 gi|14548302 Ubiquinol-cytochrome-c reductase complex core protein 0.9288 1.0355 1.00825 gi|127741 Myosin heavy chain, cardiac muscle alpha isoform (MyH 0.8016 0.907 0.86326 gi|21903382 ADP,ATP carrier protein, heart/skeletal muscle isoform 0.9794 0.9418 1.08487 gi|125313 Creatine kinase, sarcomeric mitochondrial precursor (S- 0.941 0.9892 0.92148 gi|29427692 Mitochondrial inner membrane protein (Mitofilin) 0.9584 1.1019 1.09659 gi|47117271 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitoc 0.9567 1.0813 1.039
10 gi|25089776 ATP synthase D chain, mitochondrial 1.045 0.9008 0.936611 gi|12643614 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 0.9855 1.055 0.796612 gi|14548301 Ubiquinol-cytochrome-c reductase complex core protein 0.977 1.0643 1.028713 gi|32363401 NADH-ubiquinone oxidoreductase 39 kDa subunit, mitoc 0.9544 0.9841 0.874914 gi|59799337 Carnitine O-palmitoyltransferase I, mitochondrial muscle 0.9734 0.8598 0.97615 gi|122513 Hemoglobin beta-1 chain (B1) (Hemoglobin beta-major c 1.2587 0.8148 1.326216 gi|52782785 Succinate dehydrogenase [ubiquinone] flavoprotein subu 0.9795 0.8802 0.772917 gi|51702234 Cytochrome c, somatic 0.8554 0.855 0.871218 gi|62510804 NADH-ubiquinone oxidoreductase 51 kDa subunit, mitoc 0.9999 1.1336 1.109919 gi|10720404 Voltage-dependent anion-selective channel protein 1 (VD 0.9558 0.8763 0.927620 gi|21759257 Phosphate carrier protein, mitochondrial precursor (PTP 0.9284 0.9522 0.974321 gi|2494234 Trifunctional enzyme alpha subunit, mitochondrial precu 0.9429 0.9185 0.821622 gi|47605546 ATP synthase oligomycin sensitivity conferral protein, m 1.0661 1.2229 1.178823 gi|122385 Hemoglobin alpha chain 1.4621 0.9395 1.360924 gi|52000730 Electron transfer flavoprotein-ubiquinone oxidoreductase 1.0063 0.9472 1.068825 gi|46397780 Voltage-dependent anion-selective channel protein 2 (VD 0.994 0.7796 0.924926 gi|135025 Succinyl-CoA ligase [GDP-forming] alpha-chain, mitoch 1.1187 1.0015 0.997727 gi|6648069 Acyl-CoA dehydrogenase, very-long-chain specific, mito 1.2454 1.4535 1.306828 gi|21263432 ATP synthase gamma chain, mitochondrial precursor 0.966 0.9831 1.014529 gi|51316075 Trifunctional enzyme beta subunit, mitochondrial precurs 1.0348 1.0581 1.052730 gi|2497985 Mitochondrial 2-oxoglutarate/malate carrier protein (OGC 0.9901 0.9518 0.856431 gi|136429 Trypsin precursor 0.936 0.9008 0.803232 gi|21362402 Succinate dehydrogenase cytochrome b560 subunit, mi 0.9899 1.2713 1.415333 gi|52000877 Ubiquinol-cytochrome c reductase iron-sulfur subunit, m 1.1465 0.9537 1.062234 gi|416827 Cytochrome c oxidase subunit IV isoform 1, mitochondr 0.9521 1.063 1.270135 gi|47117166 NADH-ubiquinone oxidoreductase subunit B17.2 (Comp 0.9159 1.0694 0.956736 gi|23503090 NADH-ubiquinone oxidoreductase MLRQ subunit (Comp 0.8453 0.8299 0.83
37 gi|128751 NADH-ubiquinone oxidoreductase chain 4 (NADH dehyd 1.0998 1.3008 1.177938 gi|127676 Myoglobin 1.0821 1.0059 0.815539 gi|729927 Long-chain-fatty-acid--CoA ligase 1 (Long-chain acyl-Co 0.9547 0.9015 0.903640 gi|114788 Band 3 anion transport protein (Anion exchange protein 0.9805 0.7817 1.082141 gi|3023546 Cytochrome c oxidase polypeptide VIb (Cytochrome c o 1.0534 1.1336 1.027942 gi|8928228 NDRG2 protein (Ndr2 protein) 1.1141 1.2381 0.920943 gi|20454828 ATP synthase B chain, mitochondrial precursor 0.9317 0.9631 1.063944 gi|117757 Cytochrome c1, heme protein, mitochondrial (Cytochrom 1.0053 1.0109 0.980645 gi|23503049 SAM50-like protein CGI-51 0.9398 1.0085 1.052846 gi|47117273 NADH-ubiquinone oxidoreductase 49 kDa subunit, mitoc 1.125 1.2326 1.197247 gi|1708406 Isocitrate dehydrogenase [NADP], mitochondrial precurs 1.0033 1.1002 0.996848 gi|48428148 Cytochrome c oxidase polypeptide VIc 0.9961 0.9779 0.907749 gi|47117296 NADH-ubiquinone oxidoreductase subunit B14.5b (Com 0.9141 0.9153 0.855650 gi|6166595 Myosin-binding protein C, cardiac-type (Cardiac MyBP-C 0.7116 1.0161 1.218951 gi|12643945 Voltage-dependent anion-selective channel protein 3 (VD 0.9371 0.6087 0.806452 gi|461587 ATP synthase e chain, mitochondrial 0.9164 0.9197 0.9153 gi|116982 Cytochrome c oxidase subunit 1 (Cytochrome c oxidase 1.0794 1.6895 1.685854 gi|32363396 NADH-ubiquinone oxidoreductase B8 subunit (Complex 0.9946 1.1637 0.999855 gi|128789 NADH-ubiquinone oxidoreductase chain 5 (NADH dehyd 1.1626 1.971 1.99356 gi|17380333 Ubiquinol-cytochrome c reductase complex 14 kDa prot 0.8577 0.812 0.861757 gi|23396786 NADH-ubiquinone oxidoreductase 30 kDa subunit, mitoc 0.7506 0.8655 0.672958 gi|51701449 Succinate dehydrogenase [ubiquinone] iron-sulfur protei 0.9662 0.9876 1.033359 gi|20140777 Ubiquinol-cytochrome c reductase complex ubiquinone- 0.8723 0.8361 0.941560 gi|117104 Cytochrome c oxidase polypeptide Vb, mitochondrial pr 1.0632 1.0278 1.042461 gi|52782750 ATP synthase g chain, mitochondrial (ATPase subunit G 0.879 0.6913 0.868662 gi|117100 Cytochrome c oxidase polypeptide Va, mitochondrial pr 1.0756 1.0307 0.978863 gi|20139147 NADH-ubiquinone oxidoreductase B9 subunit (Complex 0.9805 0.8983 1.004564 gi|32363395 NADH-ubiquinone oxidoreductase 13 kDa-B subunit (Co 1.052 0.9504 1.06865 gi|135762 3-ketoacyl-CoA thiolase, mitochondrial (Beta-ketothiolas 1.4354 1.5099 1.579466 gi|17380523 Spectrin alpha chain, erythrocyte (Erythroid alpha-spect 0.9823 0.7433 0.964567 gi|25108876 D-beta-hydroxybutyrate dehydrogenase, mitochondrial p 0.7651 1.1896 1.268468 gi|32363438 NADH-ubiquinone oxidoreductase subunit B14.5a (Com 1.0773 1.1949 1.059869 gi|1171862 NADH-ubiquinone oxidoreductase 23 kDa subunit, mitoc 1.1473 1.274 1.182670 gi|32363403 NADH-ubiquinone oxidoreductase PDSW subunit (Comp 1.1114 1.0316 0.956171 gi|47117291 NADH-ubiquinone oxidoreductase 15 kDa subunit (Com 1.0061 1.0209 1.045272 gi|32363402 NADH-ubiquinone oxidoreductase 19 kDa subunit (Com 0.9934 0.9451 0.889373 gi|3023355 ATP synthase epsilon chain, mitochondrial 1.012 1.336 1.2899
74 gi|117066 Cytochrome c oxidase subunit 3 (Cytochrome c oxidase 1.0425 1.1063 1.017875 gi|54038837 Prohibitin (B-cell receptor associated protein 32) (BAP 3 0.9002 1.245 1.337776 gi|2506246 Spectrin beta chain, erythrocyte (Beta-I spectrin) 1.0932 0.9099 0.884177 gi|21363012 Serotransferrin precursor (Transferrin) (Siderophilin) (Bet 1.433 1.4048 1.671178 gi|17380131 NipSnap2 protein (Glioblastoma amplified sequence) 1.0592 1.1662 0.803879 gi|21759002 Acyl carrier protein, mitochondrial precursor (ACP) (NAD 0.9253 0.8967 1.09180 gi|47605479 Calcium-binding mitochondrial carrier protein Aralar1 (M 1.142 1.3063 1.288981 gi|112985 Aspartate aminotransferase, mitochondrial precursor (Tr 1.1249 0.9788 0.868582 gi|3287881 NADH-ubiquinone oxidoreductase 18 kDa subunit, mitoc 0.9829 0.935 1.19683 gi|32363398 NADH-ubiquinone oxidoreductase B14 subunit (Complex 0.9959 0.8353 0.880384 gi|3023547 Cytochrome c oxidase polypeptide VIIa-heart, mitochon 1.1378 1.1193 1.039685 gi|24638337 Vesicle-associated membrane protein-associated protei 1.0706 1.0453 1.090486 gi|46396884 Riboflavin biosynthesis protein ribD 0.8063 0.9453 0.850487 gi|47117308 NADH-ubiquinone oxidoreductase ASHI subunit, mitoch 0.8017 0.8672 0.888888 gi|20139062 NADH-ubiquinone oxidoreductase B16.6 subunit (Comp 0.856 1.1778 0.843889 gi|114392 Sodium/potassium-transporting ATPase beta-1 chain (S 1.1966 1.5938 0.908890 gi|13431757 Programmed cell death protein 8, mitochondrial precurs 0.8394 1.0086 1.087891 gi|122646 Hemoglobin beta chain 1.3944 0.8233 1.2592 gi|122717 Hemoglobin delta chain 1.3224 0.9568 1.19193 gi|122680 Hemoglobin beta chain 1.1086 0.6969 0.52294 gi|113018 Acyl-CoA dehydrogenase, medium-chain specific, mitoc 1.1897 1.0182 0.735895 gi|24638340 Vesicle-associated membrane protein-associated protei 0.8975 1.0945 1.271896 gi|47116757 Glutathione S-transferase kappa 1 (GST 13-13) (Glutath 1.13 1.7755 1.31197 gi|52000840 NADH-ubiquinone oxidoreductase subunit B14.7 (Comp 1.1563 1.1113 1.00998 gi|20455499 NADH-ubiquinone oxidoreductase 24 kDa subunit, mitoc 0.7512 0.8399 0.712899 gi|62510510 Coiled-coil-helix-coiled-coil-helix domain containing prote 0.8996 0.9177 0.7529
100 gi|117102 Cytochrome c oxidase polypeptide Vb (VI) 1.0345 1.248 0.7659101 gi|2506313 Cytochrome c oxidase polypeptide VIa-heart, mitochond 1.4353 1.6053 1.3916102 gi|32469593 Acyl-CoA dehydrogenase family member 9, mitochondri 0.9931 0.831 0.8777103 gi|117708 Gap junction alpha-1 protein (Connexin 43) (Cx43) (Gap 0.7444 0.8554 0.5425104 gi|1709409 NADH-ubiquinone oxidoreductase 13 kDa-A subunit, mi 1.1284 0.9746 1.0065105 gi|3123245 NADH-ubiquinone oxidoreductase 24 kDa subunit 1.0779 1.0245 1.0817106 gi|117502 Calreticulin precursor (CRP55) (Calregulin) (HACBP) (ER 0.9547 1.1397 1.2674107 gi|13633877 Reticulon protein 2 (Neuroendocrine-specific protein-like 0.8207 0.9 0.9205108 gi|127167 Myosin regulatory light chain 2, ventricular/cardiac musc 0.7507 0.8006 0.8096109 gi|37077191 Acetyl-coenzyme A synthetase (Acetate--CoA ligase) (A 1.0089 0.8299 0.5473110 gi|62510451 Calcium-binding mitochondrial carrier protein Aralar2 (M 0.9153 0.9638 0.9616
22
+TOF Product (594.4): Experiment 4, 30.262 min from 050822_VanEyk_KL_fract...a=3.56509510749935590e-004, t0=-4.74340876995043890e+001
Max. 120.0 counts.
100 200 300 400 500 600 700 800 900 1000m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
120
Inte
nsity
, cou
nts
145.1054
594.3456
500.7791114.1031
291.2030
244.1644
216.1776 472.2535
585.3454175.1108 357.2324 616.3070 716.3780 944.5509
+TOF Product (594.4): Experiment 4, 30.262 min from 050822_VanEyk_KL_fract...a=3.56509510749935590e-004, t0=-4.74340876995043890e+001
Max. 120.0 counts.
113.0 114.0 115.0 116.0 117.0 118.0m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
Inte
nsity
, cou
nts
115.0999114.1031
117.1097
116.1068
Protein: ATP SYNTHASE BETA CHAIN, MITOCHONDRIAL PRECURSOR
Peptide: VLDSGAPIJ
Fragmentation Spectrum Expanded Reporter Region
No Change
P P M M
P = PlaceboM = Morphine
+TOF Product (458.5): Experiment 2, 52.128 min from 050822_VanEyk_KL_fracti...a=3.56509510749935590e-004, t0=-4.74340876995043890e+001
Max. 89.0 counts.
113.0 114.0 115.0 116.0 117.0 118.0m/z, amu
0
5
10
15
20
25
30
Inte
nsity
, cou
nts
116.1047
117.1090
115.1040114.1061
Protein: Cytochrome c oxidase subunit 1
Peptide: VFSWLATLHGGNIJ
Fragmentation Spectrum Expanded Reporter Region
Up Regulated!
+TOF Product (458.5): Experiment 2, 51.989 min from 050822_VanEyk_KL_fracti...a=3.56509510749935590e-004, t0=-4.74340876995043890e+001
Max. 58.0 counts.
100 200 300 400 500 600 700 800 900 1000m/z, amu
0
5
10
15
20
25
30
35
40
45
50
5558
Inte
nsity
, cou
nts
159.0885 527.7857
291.2032385.2246
116.1041
318.6840145.1031
492.2710460.2365
584.3385332.6849478.2550
195.0678 632.3573309.6799
P P M M
P = PlaceboM = Morphine
18O-Labeling
Compares 2 samples
Labels C-terminus of peptides
Sample complexity limited (due to 18O exchange with water over time)
Quantify from intact peptides(MS)
No steps added to protein identification
No Analysis Software available
iTRAQ
Compares up to 4 samples
Labels N-terminus + primary amines
Complex samples (can fractionate with SCX chromatography after labeling)
Quantify from fragmenting peptides (MS/MS)
Adds one step to protein identification
Analysis Software available
iTRAQ vs. 18O-Labeling
23
Topics
Protein identification by mass spectrometry
Differential protein expression techniques
Sample preparationSample preparation
Sample Preparation is Everything!
BeforeBuffer exchange
andConcentration
AfterBuffer exchange
andConcentration
Lung Lavage
Sample Preparation is Everything!
BeforeBuffer exchange
andConcentration
Serum after
removing albumin
AfterBuffer exchange
andConcentration
24
Effect of Desalting on Mass Spectrometry
1000 1500 2000 2500 3000 3500 4000 4500 Mass (m/z)
After Desalting
Protein digest in Phosphate/NaCl/DTT/urea buffer
Buffer exchange techniques
• Dialysis
• Spin dialysis
• Chromatography
• Precipitation/resuspension
• Slow
• Detergents can concentrate with protein
• Protein losses
• Difficult to resolubilize, protein losses
Detect Low Abundance Proteinsby
Fractionating Proteins Before Labeling
Bergh et al. 2003Electrophoresis24: 1471-1481
AdultCat
Kitten30 day
GradientC18 column
25
Detecting Low Abundance Proteins by DIGE Analysis
Total Lysates
40% Acn Fraction 46% Acn Fraction
Kitten Cy3Adult Cy5
pI3.0 11.0
250150
75
50
37
25
10
10.53.5 5.8 8.2
15 ug protein, 7 cm IPG strip,1 gel
75 ug protein, 24 cm IPG strip,3 gels
MS Compatible Protein Stains
Silver stains - no gluteraldehyde fixation stepMany protocolsCheck with MS lab
Colloidal Coomassie blue stain (preferred)SimplyBlue (Invitrogen) GelCode Blue (Pierce) (Electrophoresis 9, 255-262, 1988)
Fluorescent stainsSyproRuby (Molecular probes)Deep Purple (GE Healthcare)
26
Differential Protein ExpressionProtein Pre-Fractionation (sample complexity)
(low abundance proteins)Protein/Peptide Labeling
Fluorescent tag (Cy dyes)Stable isotope (13C, 15N, 18O)Mass tags (Signature ions)
Protein/Peptide SeparationGel Electrophoresis (MW, pI)Chromatography (MW, charge, affinity)Both
Quantification (comparison of label)
Protein Identification (by mass spectrometry)
Best Approach?There is NO one best approach
Gel based techniques - Total Protein(isoforms, PTMs)
MS based techniques- Peptides(membrane proteins, large or small proteins)
All approaches are complementary
Technically challenging
