Upload
espen
View
78
Download
0
Embed Size (px)
DESCRIPTION
PAF introduction. The PAF (also called UNIL Proteomics Platform) is a research and service core facility devoted to the high-performance analysis of proteins. Service : Identification of gel-separated proteins by MS Project discussion - PowerPoint PPT Presentation
Citation preview
PAF introduction
The PAF (also called UNIL Proteomics Platform) is a research and service core facility devoted to the high-performance analysis of proteins
•Service :
•Identification of gel-separated proteins by MS
•Project discussion
•2D-PAGE and/or other multi-dimensional separation techniques
Research :
•Development of proteomics methods
•Applications to biological problems
•Teaching :
•Techniques, possibilities & limitations of proteomics approaches
PAF technology and instrumentation
Nano-HPLC- Quadrupole-time-of-flightMass spectrometer
Nano-HPLC-triple quadrupole ion trapMass spectrometer
2D-electrophoresis1D-electrophoresis
260404RPChistonesFRAC001:1_UV1_280nm 260404RPChistonesFRAC001:1_Conc 260404RPChistonesFRAC001:1_Fractions 260404RPChistonesFRAC001:1_Inject
-30.0
-20.0
-10.0
0.0
10.0
20.0
30.0
mAU
0.0 5.0 10.0 15.0 20.0 25.0 ml
A1 A2 A3 A4 A5 A6 A7 A8 A9A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 D12 D11 D10 D9
Liquid Chromatography
New : access to ABI 4700 TOF/TOF™ (courtesy Dept. Biochemistry)
MALDI - Tandem Time of Flight Mass Spectrometer for High Throughput Protein identification
Challenges of in vivo proteomics
• Complexity : – 35’000 genes (?) in H. sapiens, – 15’000 expressed in a single cell ? – but >50’000 chemically different protein species ?
• Dynamic range :– 105 x or 106 x between low and high-abundance proteins
• Plasticity : – continuous variation in protein expression pattern (every s),
PTM’s, degradation,…
PTM analysisPTM analysis
Interaction / Functional Interaction / Functional ProteomicsProteomics
Protein expression Protein expression analysisanalysis
FOCUS : Subcellular fractionOrganelleProtein Complex20-200 proteins
FOCUS : Complex samplesWhole proteomes200 and more proteins
FOCUS :Single protein
1-20 proteins
Sample complexity
Analytical Detail
Overview : classes of proteomics experiments
Proteomics
• 2D-PAGE
• Mass Spectrometry for proteomics
• Proteomics workflows and applications
2D-PAGE
How to create a pH gradient ?
IEF: the principle
Improvements in 2D-PAGEImprovements in 2D-PAGE
-+
- IPG (Immobilised Ph Gradient) strips for the first dimensionpH-forming chemical groups are grafted onto the
polyacrylamide matrix, creating a mechanically stable pH gradient
+ + mechanical stability+ + reproducibility+ + loadable amounts+ + „zoom“ pI ranges
pH 3.0 10.0
Equilibration step 1 : •SDS•Buffer pH 6.8•DTT (reduce –S-S-)
Equilibration step 2 : •SDS•Buffer pH 6.8•Iodoacetamide (alkylate –S-S-)
After IEF : equilibration and 2nd dimension
+ -
6.5 10 3.0 5.5 8.5
pI
100
75
50
37
2010
150
Database of 2D images with clickable spots :
www.expasy.org/ch2d/
One protein many spots
Post-translational modifications can result in changes in pI and/or MW
Spot “trains” for extensively modified proteinsCaused by
GlycosylationPhosphorylationAcetylation (K)…
2D-PAGE and image analysis are used for studying changes in composition of the
proteome
Control
Stimulus applied
•Spot detection
•Spot quantification
•Gel-to-gel•Matching
•Presence/absenceof spot
•Up/down regulation
•Statistic analysis
Software-based image analysis
Mass Spectrometry in proteomics
Mass spectrometry : essential functions
SAMPLEION
GENERATIONION
SEPARATIONION
DETECTION
ION SOURCE MASS ANALYZER DETECTOR
ESI : Electrospray Ionisation
MALDI : Matrix Assisted Laser Disorption/Ionization
QuadrupolesIon trapsTime-of-flight with reflectronTOF/TOFFT-ICR (Fourier transform –Ion Cyclotron Resonance)
Faraday cupScintillation counterElectromultiplierHigh-energy dynodes with electronmultiplierArray (detector)FT-MS
Masses and mass measurements
•All mass spectrometers function measure molecules in their ionized state
•All values determined by MS are relative to the m/z assumed by the molecule after the ionization process
The relationship between the molecular mass (m) and the m/z value can be calculated as follows:
m/z = (m + (mA * z )) / z
mA is the mass of the adduct responsible for ionization (typically H+ for positive MS mode).
MALDI (Matrix Assisted Laser Desoprtion Ionisation
MALDI TOF (Time Of Flight)
MALDI IONISATIONMALDI IONISATION
• Great for Peptide Mass Fingerprinting– Fast– Easy to measure – Sensitive– Salt-tolerant (to some extent)– Also good for larger MW (small proteins)– Sample on a stable support (no time constraints)– 1+ ions simpler data analysis
•High accuracy needs careful calibration•Difficult (but possible) to do MS/MS by MALDI•Signal suppression in complex mixtures•Crystallisation conditions influence results
disadvantages
MALDI TOFMALDI TOF
MALDI-TOF of a tryptic digest of BSA
+TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ...a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded
Max. 1305.0 counts.
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
Inte
ns
ity, c
ou
nts
927.59
847.59
1440.00 1479.98 1567.94
1640.161022.56869.07
1305.871163.77 1481.98
1296.861249.771050.55871.07 1024.56 1417.931283.91789.53 1142.86 1443.011073.03 1595.951386.76 1824.09857.14
1108.71 1292.95978.60 1790.101501.84 1616.92
YLYEIAR
LSQKFPK LVNELTEFAK
FKDLGEEHFK
HPEYAVSVLLR
HLVDEPQNLIK
LGEYGFQNALIVR
KVPQVSTPTLVEVSR
DAFLGSFLYEYSR
??
?
? ??
P. Kebarle, M. Peschke / Analytica Chimica Acta 406 (2000) 11–35
• molecules compete for ionisatione.g. Na+ >> Peptide
ELECTROSPRAY IONISATIONELECTROSPRAY IONISATION
• Great for MS/MS– Can be directly coupled to reversed phase LC
(separation !)– Sensitive– Excellent for MS/MS due to 2+/3+ ions
•Sample introduction more complex•Data analysis more difficult (2+/3+ ions) •one-shot sample analysis (time constraints)•Very low tolerance to contaminants
disadvantages
ELECTROSPRAY IONISATIONELECTROSPRAY IONISATION
1+ versus 2+ ions
1160 1162 1164 1166 1168 1170 1172 1174m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
8084
Intensity, counts
1163.76
1164.76
1165.74
1166.781162.70
m=1.0 Da
1.0 Da
1.0 Da
1+
580.0 581.0 582.0 583.0 584.0 585.0 586.0m/z, amu
0
20
40
60
80
100
120
140
160
180
200
Intensity, counts
582.28
582.77
583.27
m=0.5 Da
0.5 Da
0.5 Da
2+
Modes of measurement : MS & MS/MS
•Ion production (ionisation)
•Ion separation
•Ion detection
•Ion production (ionisation)
•Ion separation – isolation of “parent” ion
•Ion fragmentation (CID)
•Ion separation – separate fragment ions
•Ion detection - measure fragment ions
MS
Tandem MSMS/MS
Tandem MS (MS/MS) facts
• MS/MS results in the acquisition of pseudo-sequence information for multiple (as many as possible) tryptic peptides in addition to intact peptide mass information
• MS/MS needs an instrument able to perform ion isolation and fragmentation, in addition to regular MS
• MS/MS results in the acquisition of multiple orthogonal data files (1 spectrum / peptide)
•CID= collision induced dissociation
•Low energy (<100 eV) vs high energy collisions (>> 100 eV)
•Precursor ion = parent ion : the one being fragmented
•Daughter ions = fragment ions produced by CID
•Tandem mass spectrometry = MS/MS•- here : the combination of ion selection / CID / fragment analysis
•ESI of tryptic peptides typically generates doubly charged ions due to the presence of Lys or Arg at the C terminal end of the peptides
•y and b-ion series fragments are usually observed in MS/MS fragmentation spectra.
•CID= collision induced dissociation
•Low energy (<100 eV) vs high energy collisions (>> 100 eV)
•Precursor ion = parent ion : the one being fragmented
•Daughter ions = fragment ions produced by CID
•Tandem mass spectrometry = MS/MS•- here : the combination of ion selection / CID / fragment analysis
•ESI of tryptic peptides typically generates doubly charged ions due to the presence of Lys or Arg at the C terminal end of the peptides
•y and b-ion series fragments are usually observed in MS/MS fragmentation spectra.
MS/MS Glossary and facts
Covalent bonds being broken ion series
Covalent bonds being broken ion series
ESI & Quadrupole-based instruments
Triple Quadrupole
Triple Quadrupole-Ion trap
Quadrupole-QuadrupoleTOF
Ion Trap (3D trap)
All these instruments can perform MS/MS fragmentation experiments
• Identifying proteins by mass spectrometry
MS-based protein identification : general concept
Protein sample
Protein fragments (5-30 AA peptides)
Exact masses of peptides
Fragmentation (MS/MS)spectrum of each peptide
Protein sequence(s)
Protein fragment sequences (same protease specificity)
Calculated exact masses of peptides
Calculated fragmentation spectrum of each peptide
Specific proteasee.g. trypsin
MS
software
software
software
Best Match(es)
experimental In silico
• Protein identification by Peptide Mass Fingerprinting (PMF)
+TOF MS: 50 MCA scans from Sample 1 (BSA Digest 100 fmol) of BSA Digest 100 fmol MS ...a=3.56217430068478150e-004, t0=3.64725878201043440e+001, Thresholded
Max. 1305.0 counts.
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
Inte
nsity
, co
un
ts
927.59
847.59
1440.00 1479.98 1567.94
1640.161022.56869.07
1305.871163.77 1481.98
1296.861249.771050.55871.07 1024.56 1417.931283.91789.53 1142.86 1443.011073.03 1595.951386.76 1824.09857.14
1108.71 1292.95978.60 1790.101501.84 1616.92
Extracted peak list
m/z
847.5896869.0722922.5712923.5815927.59041022.55511050.55331163.76951164.75311193.73931249.77051250.81031296.85561297.84991305.86681416.89291440.00081479.97731482.95831567.94171640.16351824.06…
Information contained in MS spectrum
Search form…
Results page…
• Protein identification by MS/MS
Experimental set-up : nanoLC-MS/MS
database correlation
C18 ColumnL = 10 cmID = 50-100 µm
Mass spectrometer
T-splitter
~5%
HPLCpumps
~95%further analysis
(waste)sample
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Time, min
0.0
4000.0
8000.0
1.2e4
1.6e4
2.0e4
2.4e4
2.8e4
3.2e4
3.6e4
Intensity, cps
568.58
496.57 563.23354.11445.07
+TOF Product (461.7)
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z, amu
0
5
10
15
20
Intensity, counts
86.09
147.01
635.31260.17175.06
270.11522.23
748.39359.01
y8y7
y6
y5
y4
y3
y2y1
SSILFDIK
461.71
375.19
An on-line LC-ESI-MS experiment with automatic data acquisitionAn on-line LC-ESI-MS experiment with automatic data acquisition
Mr peptide : 921.4
MeCNgradient
25 30 35 40 45 50 55 60 65 70 75 80Time, min0.00
5.00e4
1.00e5421.68
653.32464.17 582.25
722.28 501.73507.75
740.41595.29 700.34
Chromatogram : Total ion current vs. time
MS/MS peptide 4 : +TOF Product (662.8):
10.0
14.0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z, amu
0.0
5.0
235.08
166.06147.10
262.10
120.06409.18207.09
663.3252110.04
614.2136
138.06
Full scan : +TOF MS:
m/z, amu
400 450 500 550 600 650 700 750 800 850 9000
200
400518.82
777.8176536.80
662.76 531.47
507.1541
1553 (2+)
1553 (3+)1
2
34
Automated LC- MS/MS run
+TOF Product (653.3)
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z, amu
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.019.7
Intensity, counts
251.12
110.06
223.13
138.05 653.36
332.21
594.2586.09
350.24 712.461055.49
373.30 566.32465.16 956.41 1168.56166.05 841.53
206.16
a1
b1
I
a2
b2
y10
Ib3 y9
b4 y8a5
b5
y7
y6
b8
y3
Iy2
y1
Residue Immonium a b y-----------------------------------------------------------------------------------H, His 110.07 110.07 138.06 1305.71L, Leu 86.09 223.15 251.15 1168.65V, Val 72.08 322.22 350.21 1055.57D, Asp 88.03 437.25 465.24 956.50E, Glu 102.05 566.29 594.28 841.47P, Pro 70.06 663.34 691.34 712.43Q, Gln 101.07 791.40 819.39 615.38N, Asn 87.05 905.44 933.44 487.32L, Leu 86.09 1018.53 1046.52 373.28I, Ile 86.09 1131.6157 1159.61 260.19K, Lys 101.10 1259.7106 1287.70 147.11
MH22+
precursor
Matching of MS / MS data
Black : predictedRed : predicted and detected
Mascot search outputMascot search output
Mascot Search Results
Significant hits: ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). ALBU_CANFA (P49822) Serum albumin precursor (Allergen Can f 3). VWF_PIG (Q28833) Von Willebrand factor precursor (vWF) (Fragment). CIQ3_BOVIN (P58126) Voltage-gated potassium channel protein KQT-like 3 RYR2_RABIT (P30957) Ryanodine receptor 2 (Cardiac muscle-type ryanodin K2CA_BOVIN (P04263) Keratin, type II cytoskeletal 68 kDa, component IA ALFB_RABIT (P79226) Fructose-bisphosphate aldolase B (EC 4.1.2.13) (Li
ALBU_BOVIN Mass: 71244 Total score: 711 Peptides matched: 12 (P02769) Serum albumin precursor (Allergen Bos d 6). Observed Mr(expt) Mr(calc) Delta Miss Score Rank Peptide
15 461.80 921.58 921.48 0.10 0 55 1 AEFVEVTK 19 501.80 1001.58 1001.58 0.01 0 31 1 LVVSTQTALA 31 569.80 1137.58 1137.49 0.09 0 72 1 CCTESLVNR 33 582.30 1162.58 1162.62 -0.04 0 76 1 LVNELTEFAK 47 653.40 1304.78 1304.71 0.08 0 90 1 HLVDEPQNLIK 58 722.40 1442.78 1442.63 0.15 0 87 1 YICDNQDTISSK 60 739.80 1477.58 1477.52 0.07 0 43 1 ETYGDMADCCEK 61 740.40 1478.78 1478.79 -0.00 0 61 1 LGEYGFQNALIVR 64 751.90 1501.78 1501.61 0.18 0 37 1 EYEATLEECCAK 74 547.30 1638.88 1638.93 -0.05 1 85 1 KVPQVSTPTLVEVSR 97 627.70 1880.08 1879.91 0.16 0 34 1 RPCFSALTPDETYVPK 98 636.70 1907.08 1906.91 0.16 0 42 1 LFTFHADICTLPDTEK
Orthogonal datasets and confidence levels
Database : 100’000 sequences 500 spectra
Probability of one (any) spectrum “accidentally” matching a sequence (wrong match) : 1/100’000 x 500 = 5.10-3 (0.005)
Probability of 2 spectra “accidentally” matching the same sequence (wrong match) : 5.10-3 x 5.10-3 = 2.5 x 10-5
Much higher confidence of identification with at least two peptides matching the same protein sequence
Every peptide is unambiguously assigned to its “parent “ sequence, therefore many proteins can be identified in one sample during one run
PMF :
one MS spectrum one dataset (peak list)
MS/MS :
n MS/MS spectra n orthogonal datasets
m/z
time
Chromatographic Separation (reversed-phase)
Tandem mass spectra of 50-2000 peptides
Q7Z5Y2 Mass: 118789 Total score: 178 Peptides matched: 6 Rho-interacting protein 3. Mr(calc) Score Peptide 930.48 42 EGLTVQER 1032.54 11 NWIQTIMK 1206.63 29 FSLCILTPEK1369.75 24 LSTHELTSLLEK 1406.77 55 FFILYEHGLLR 1775.88 16 QVPIAPVHLSSEDGGDR
ProteasedigestionPeptideextraction
Nano-HPLC
MS/MS
Summary : Typical Analytical Workflow
Database matchesDHX9_HUMAN ATP-dependent RNA helicase ANFM_HUMAN Neurofilament triplet M proteinQ9BQG0 Hypothetical proteinMYO6_HUMAN Myosin VI. TP2A_PIG DNA topoisomerase II, alpha isozymeQ7Z5Y2 Rho-interacting protein 3. FLIH_HUMAN Flightless-I protein homolog. TP2B_MOUSE DNA topoisomerase II, beta isozymeS3B1_HUMAN Splicing factor 3B subunit Q8VCW5 Similar to alpha internexin neuronal Q8CHF9 MKIAA0376 protein (Fragment).
Protein sequence database
Database searching Software (MASCOT)Output :
•Protein identification in simple/complex mixtures
•Extensive sequence coverage and peptide mapping
•Analysis of modified peptides possible
Biological question
Caveat : Protein identification IS NOT
protein characterisation
Two peptides are enough to identify a proteinBut
We are still identifying two peptides, not the entire protein
Highly similar sequences cannot bedistinguished
For finding PTMs extensive Sequence coverage is essential !!
2 Technology, workflows and
applications : what is available
New and old tools
Protein separation techniques- Liquid chromatography- Electrophoresis -…
Protein identification techniques- Mass Spectrometry- Antibody-based techniques
Protein quantification techniques- Antibody based techniques - dye-binding techniques - Mass Spectrometry
Genome sequence databases
Protein sequence databases
Biological knowledgebases : - functions- pathways- seq. motifs- 3D structures
WORKFLOWS 1 : WORKFLOWS 1 : „classical“„classical“ 2D-PAGE 2D-PAGE
++ MALDI TOF MALDI TOF
11 22
3A3A 3B 3B 445 667 788
9910101111 1212
1
14
8
15
Workflow 1 : adaptation of bacteria to growth conditionsWorkflow 1 : adaptation of bacteria to growth conditions
Normal medium Low Glucose
Wick LM, et al, Environ Microbiol 3: 588-599, 2001
E.Coli adapts to a very low glucose medium by up- and downregulating a set of 15 proteins
M1
M2
M3
M4
M5
M6
M7
M8
Peptide Mass FingerprintingSearch with mass list
M1......M8
Tryptic digestion
Nr mw* pI* Acc. N. Name Function
1 52.2 5.07 P25553 aldA central metabolism
2 60.3 6.21 P23847 dppA peptide transport
3 47.5 5.06 P05313 aceA Central metabolism
4 48.45 6.71 P10904 ugpB transport
5 55.2 6.02 P00822 atpA proton transport/energy
6 ? ? P76108 ydcS transport
7 41.3 7.03 P02917 livJ amino acid transport
8 40.7 5.22 P02928 malE sugar transport
9 33.36 5..25 P02927 mglB sugar transport
10 61.5 5.81 P37192 gatY catabolism
11 30.9 7.76 P02925 rbsB sugar transport
12 25.8 5.22 P09551 argT amino acid transport
Workflow 1 : adaptation of bacteria to growth conditionsWorkflow 1 : adaptation of bacteria to growth conditions
Metabolic enzymes and transport proteins affected
Utilisation of alternative sources of energy
WORKFLOW 1 : quantitation and kineticsWORKFLOW 1 : quantitation and kinetics
UgpB (4)
00.020.040.060.080.100.12
1 2 3 4 5
DppA (2)
00.51.01.52.02.53.03.54.0
1 2 3 4 5
AldA (1)
00.20.40.60.81.01.21.41.6
1 2 3 4 5
AceA (3A)
0123456
1 2 3 4 5
AceA (3B)
00.20.40.60.81.01.2
1 2 3 4 5AtpA (5)
00.050.100.150.200.250.300.35
1 2 3 4 5
YdcS (6)
00.020.040.060.080.100.120.140.16
1 2 3 4 5
LivJ (7)
00.51.01.52.02.53.03.54.04.55.0
1 2 3 4 5MalE (8)
02468
101214
1 2 3 4 5
GatY (10)
00.51.01.52.02.53.0
1 2 3 4 5
RbsB (11)
00.10.20.30.40.50.60.70.80.9
1 2 3 4 5
MglB (9)
0246810
12141618
1 2 3 4 5
ArgT (12)
00.51.01.52.02.53.0
1 2 3 4 5
MalI (13)
00.050.100.150.200.25
1 2 3 4 5
Relative protein quantities as a function of time during the adaptation process
Time points : 1) Start – batch
culture2) Adapted
bacteria put back into high-glucose batch culture
3) 40 hr adaptation culture
4) 156 hr adaptation culture
5) 500 hr adaptation culture
• Ideal interface to biology• Analytical and micropreparative• Robust• Solid phase chemistry of proteins• Easy, low-tech• Removal of contaminants :
– At the loading point– After migration during fix / staining steps
Why is SDS-PAGE such a good preparation method?Why is SDS-PAGE such a good preparation method?
• protein digestion in gel: non quantitative• peptide sequence recovery: usually incomplete• whole protein recovery: poor
Disadvantages
In-gel digestion: solid phase chemistry of proteinsIn-gel digestion: solid phase chemistry of proteins
WORKFLOWS 2 : WORKFLOWS 2 :
General shotgun protein identification General shotgun protein identification techniquestechniques
example : example : Affinity pull-downAffinity pull-down
++ 1D-PAGE 1D-PAGE
++ LC-MS/MS LC-MS/MS
Shotgun sequencing from complex mixturesShotgun sequencing from complex mixtures
Denaturation, Proteolytic digestion
Db search
Multiprotein complex
List of identified proteins
1. P452182. P215433. Q125884. P326515. Q012456. ….
Complex peptide mixture(1000-20000 species)
Rp-LC-MSMS run
Alternative : MuDPIT (Multi Dimensional Protein Identification Alternative : MuDPIT (Multi Dimensional Protein Identification Technology) Technology)
Denaturation, Proteolytic digestion
Strong Cation Exchange(SCX)separation
Db search
Multiprotein mixture (complex)
List of identified proteins
1. P452182. P215433. Q125884. P326515. Q012456. ….
Complex peptide mixture(1000-20000 species)
Rp-LC-MSMS runs
Post-digestion separation of peptides by two-dimensional liquid chromatography Post-digestion separation of peptides by two-dimensional liquid chromatography instead of separation of proteinsinstead of separation of proteins
TIC: from 151002_ACO_B4strep.wiff Max. 5.3e5 cps.
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.3e5
Inte
ns
ity, c
ps
653.36
133.06
526.26
406.23303.13 852.44
203.11199.19402.54186.12
472.25541.11
86.10536.34 449.15574.661064.42 629.30
665.38494.25
330.18 527.26
615.4186.10563.30
599.32
409.18
A VERY complex mixture – direct analysis (no separation)A VERY complex mixture – direct analysis (no separation)
Mascot Search ResultsUser : MQ Email : Search title :151002_ACO_B4strep.wiff : Angelos frac B4 IP strept MS data file : C:\DOCUME~1\paf\LOCALS~1\Temp\mas5D.tmp Database : Sprot 4028 (114033 sequences; 41888693 residues) Taxonomy : Mammalia (mammals) (23838 sequences) Timestamp : 17 Oct 2002 at 08:09:30 GMT
Significant hits:
ALBU_BOVIN (P02769) Serum albumin precursor (Allergen Bos d 6). DNM1_HUMAN (P26358) DNA (cytosine-5)-methyltransferase 1 (EC 2.1.1.37) AC15_HUMAN (P35251) Activator 1 140 kDa subunit (Replication factor C IF16_HUMAN (Q16666) Gamma-interferon-inducible protein Ifi-16 (Interfe K1CJ_HUMAN (P13645) Keratin, type I cytoskeletal 10 (Cytokeratin 10) ( K22E_HUMAN (P35908) Keratin, type II cytoskeletal 2 epidermal (Cytoker ACF7_HUMAN (Q9UPN3) Actin cross-linking family protein 7 (Macrophin) ( AC14_HUMAN (P35250) Activator 1 40 kDa subunit (Replication factor C 4 ALBU_FELCA (P49064) Serum albumin precursor (Allergen Fel d 2). AC15_MOUSE (P35601) Activator 1 140 kDa subunit (Replication factor C DYHC_MOUSE (Q9JHU4) Dynein heavy chain, cytosolic (DYHC) (Cytoplasmic AC12_HUMAN (P35249) Activator 1 37 kDa subunit (Replication factor C 3 EF11_CRIGR (P20001) Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elonga RYR3_HUMAN (Q15413) Ryanodine receptor 3 (Brain-type ryanodine recepto K2C1_HUMAN (P04264) Keratin, type II cytoskeletal 1 (Cytokeratin 1) (K PLE1_RAT (P30427) Plectin 1 (PLTN) (PCN). AHNK_HUMAN (Q09666) Neuroblast differentiation associated protein AHNA TRYP_PIG (P00761) Trypsin precursor (EC 3.4.21.4). ACF7_MOUSE (Q9QXZ0) Actin cross-linking family protein 7 (Microtubule CENF_HUMAN (P49454) CENP-F kinetochore protein (Centromere protein F) ALBU_HUMAN (P02768) Serum albumin precursor. PLE1_HUMAN (Q15149) Plectin 1 (PLTN) (PCN) (Hemidesmosomal protein 1) TRI4_HUMAN (Q15650) Activating signal cointegrator 1 (ASC-1) (Thyroid NF1_HUMAN (P21359) Neurofibromin (Neurofibromatosis-related protein N NEBU_HUMAN (P20929) Nebulin
. . .
A VERY complex mixture still gives results, but...A VERY complex mixture still gives results, but...
450 500 550 600 650 700 750 800 850 900
m/z, amu
01020
40
60
80
100
120
140
160
180
Intensity, counts
18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
Time, min
0.02.0e44.0e46.0e48.0e41.0e51.2e51.4e51.6e51.8e52.0e52.2e52.4e52.6e52.7e5
Intensity, cps
120.08157.15
545.75581.28545.75
652.36216.11681.35
670.84131.09 343.18498.55 738.39555.22 153.08561.29569.27 548.74 499.72489.53 445.07 499.70 469.25469.24 651.85
445.10 445.09 648.38
........ how deep are we going ?........ how deep are we going ?
analyzedmissed
some protein prefractionation
is necessary !
TNF family of ligands and TNF-receptor familyTNF family of ligands and TNF-receptor family
Bodmer JL et al, TIBS. 2002 27(1):19-26
(-)FasL
(+)FasL
FADD
Casp.8
Fas
Analysis of apoptotic signalling complexesAnalysis of apoptotic signalling complexes
Real lifeModel
The Fas (CD95) signalling complex (DISC)
Flip
Casp.10
Fc-FasL
?
?
Western blot
(-)BAFF
(+)FasL
Analysis of apoptotic signalling complexes : negative controlAnalysis of apoptotic signalling complexes : negative control
* run 1 cm* no fix, no stain !* cut, digest
1 cm
(-)BAFF
(+)FasL
LCMS runs Fas/Baff
TIC: from 180702BaffHL.wiff Max. 3.8e5 cps.
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
Inte
ns
ity, c
ps
TIC: from FAS_180702_HL.wiff Max. 4.0e5 cps.
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
Inte
ns
ity, c
ps
603.33
655.82
328.19
130.11
405.33
337.17
864.45
171.17582.29
643.87 603.69
158.06186.16
171.18 607.38120.11620.67449.15
BAFF complexHL sample
FasL complexHL sample
600 spectra
680 spectra
LC-MS/MS ANALYSISLC-MS/MS ANALYSISBAFF HL search resultsDatabase : MSDB 200402 (851746 sequences; 265326103 residues)Taxonomy : Mammalia (mammals) (166849 sequences)
Significant hits: Q96RF8 SSA1.- Homo sapiens (Human). BAB27292 AK010960 NID: - Mus musculus K2C1_HUMAN Keratin, type II cytoskeletal 1 (Cytokeratin 1) A45935 dnaK-type molecular chaperone hsc70 - mouse KRHU0 keratin 10, type I, cytoskeletal - human C25437 tubulin beta-3 chain - mouse Q9CWA2 ATP SYNTHASE, H+ TRANSPORTING MITOCHONDRIAL F1 CAA30026 HSHA44G NID: - Homo sapiens CAB59134 SEQUENCE 1 FROM PATENT WO9818921 PRECURSOR A44861 keratin, 67K type II epidermal - human Q9BWB7 HEAT SHOCK 70KD PROTEIN 9B (MORTALIN-) (Human). A26168 ribophorin I precursor - human PT0207 Ig gamma chain C region - chimpanzee PWHUA H+-transporting two-sector ATPase alpha chain precursor - human JC1473 H+-transporting ATP synthase (EC 3.6.1.34) alpha chain - mouse I77403 tubulin alpha-1 chain - human AAA57233 MUSHP7A2 NID: - Mus musculus CAA82315 HSKERAT9 NID: - Homo sapiens A29904 keratin 5, type II, epidermal - human HHHU84 heat shock protein 90-beta [validated] - human AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN .-(Human). B26168 ribophorin II precursor - human CAA34756 HSEF1AC NID: - Homo sapiens S21097 alpha-1-antitrypsin precursor - bovine S04652 Ca2+-transporting ATPase (EC 3.6.1.38) 2, - pig CAA41735 BTBSA NID: - Bos taurus Q9UK02 BIP PROTEIN (FRAGMENT).- Homo sapiens (Human). KRHUEA keratin 6a, type II - human A22224 actin alpha, vascular smooth muscle - mouse Q96GA6 UNKNOWN (PROTEIN FOR MGC:15420).- Homo sapiens (Human). Q8WZ42 TITIN.- Homo sapiens (Human). I84741 RNA helicase - mouse Q9TS10 78 KDA APAMIN BINDING PROTEIN.- Bos taurus (Bovine). AAH02690 BC002690 NID: - Homo sapiens I48385 RNA helicase TNZ2 - mouse Q96FZ6 HEAT SHOCK 60KD PROTEIN 1 (CHAPERONIN).(Human). AAA56753 HSU15637 NID: - Homo sapiens CAA58470 HSPXMP11 NID: - Homo sapiens JQ0028 cytokeratin 19 – mouse…………………………………
FasL HL search results
Database : MSDB 200402 (851746 sequences; 265326103 residues)Taxonomy : Mammalia (mammals) (166849 sequences)
Significant hits: A37241 52K autoantigen Ro/SS-A - human Q8WUC1 TUBULIN, BETA 5.- Homo sapiens (Human). C25437 tubulin beta-3 chain - mouse CAC39526 SEQUENCE 15 FROM PATENT WO0129232.-HUman AAH19046 SIMILAR TO IMMUNOGLOBULIN HEAVY CONSTANT GAMMA 3 K1CJ_HUMAN Keratin, type I cytoskeletal 10 (Cytokeratin 10-(Human). I37383 FAS soluble protein - human A24903 tubulin alpha-1 chain - Chinese hamster A44861 keratin, 67K type II epidermal - human I38707 Fas ligand - human AAG41947 AF304164 NID: - Homo sapiens Q9BDN1 CD95L PROTEIN.- Cercocebus torquatus atys CAA82315 HSKERAT9 NID: - Homo sapiens AAB86467 IMMUNOGLOBULIN GAMMA HEAVY CHAIN (Human). NUCL_HUMAN Nucleolin (Protein C23).- (Human). 1ATS heat shock cognate protein 70 kD (44 kD chaperone ATPase 1FC1A Ig gamma-1 chain C region (Fc fragment), chain A - human I61769 keratin 6d, type II - human (fragment) A29904 keratin 5, type II, epidermal - human A40389 translation elongation factor eEF-1 alpha chain (clone pS1) - rat AAB46730 HSU86214 NID: - Homo sapiens CAD23746 IMMUNOGLOBULIN GAMMA HEAVY CHAIN CONSTANT REGION Q10466 TITIN, HEART ISOFORM N2-B (EC 2.7.1.-) (CONNECTIN).-(Human). Q8WZ42 TITIN.- Homo sapiens (Human). 1D3OA trypsin (EC 3.4.21.4), chain A - pig CAC20457 IMMUNOGLOBULIN HEAVY CHAIN CONSTANT GAMMA 4.- (Human). O75634 HEAT SHOCK PROTEIN 70 TESTIS VARIANT.- (Human). Q8WTZ6 RIBOSOMAL PROTEIN L18.- Homo sapiens (Human). 1NBMC f1-atpase (EC 3.6.1.34) delta and 1 epsilon subunits, chain C - bovine Q96PE2 TUMOR ENDOTHELIAL MARKER 4.- Homo sapiens (Human). Q9R1Q3 GLIAL FIBRILLARY ACIDIC PROTEIN ALPHA.- Rattus norvegicus (Rat). ITSH alpha-1-antitrypsin precursor - sheep CAA27396 MMACTBR2 NID: - Mus musculus Q8WXH0 NUANCE.- Homo sapiens (Human). Q9GL40 FAS ANTIGEN.- Macaca mulatta (Rhesus macaque). A33370 H+-transporting ATP synthase beta chain precursor, mitochondrial CAB76567 MMU250841 NID: - Mus musculus Q9GK28 FAS ANTIGEN APO-1/CD95.- Macaca arctoides (Stump-tailed macaque). Q9BZL4 MYOSIN BINDING SUBUNIT 85.- Homo sapiens (Human).…………………………………
Database search 1. Protein 1
2. Protein 23. Protein 34. …Database
search
Database search
1. Protein 12. Protein 23. Protein 34. …1. Protein 12. Protein 23. Protein 34. …
1. Protein 12. Protein 23. Protein 34. Protein 45. Protein 56. Protein 67. …
Total list sample 1
1. Protein 12. Protein 23. Protein 34. Protein 45. Protein 56. Protein 67. …
Total list sample 2
1. Protein 12. Protein 23. Protein 34. Protein 45. Protein 56. Protein 67. …
Total list sample 1
1) COMMON CONTAMINANTS ( PROTEINS STICKING TO BEADS)
2) LIGAND-COPURIFYING PROTEINS (EX SJOGREN SYNDROME 52 KDA)
3) COMMON HITS (WHAT IS IN BOTH LISTS)
Subtract from each list :
FILTERED RESULTSFILTERED RESULTS
FILTERED RESULTSFILTERED RESULTS
MASCOT DATABASE SEARCH Fas (CD95) complex
MASCOT DATABASE SEARCHBAFF receptor complex
ICE8_HUMAN (Q14790) Caspase-8
RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen
RO52_HUMAN (P19474) 52 kDa Ro protein (Sjogren syndrome type A antigen
RS3_HUMAN (P23396) 40S ribosomal protein S3 RS3_HUMAN (P23396) 40S ribosomal protein S3
FADD_HUMAN (Q13158) FADD
K2C1_HUMAN (P04264) Keratin K2C1_HUMAN (P04264) Keratin
GC1_HUMAN (P01857) Ig gamma-1 chain C region GC1_HUMAN (P01857) Ig gamma-1 chain C region
CFLA_HUMAN (O15519) CASP8 and FADD-like apoptosis regulator
RS8_HUMAN (P09058) 40S ribosomal protein S8
GBLP_HUMAN (P25388) Guanine nucleotide-binding protein
GBLP_HUMAN (P25388) Guanine nucleotide-binding protein
TNR6_HUMAN (P25445) TNF-family receptor CD95
RL7A_MOUSE (P12970) 60S ribosomal protein L7a RL7A_MOUSE (P12970) 60S ribosomal protein L7a
RL7_HUMAN (P18124) 60S ribosomal protein L7 RL7_HUMAN (P18124) 60S ribosomal protein L7
ICEA_HUMAN (Q92851) Caspase-10
PHB_HUMAN (P35232) Prohibitin
K22E_HUMAN (P35908) Keratin K22E_HUMAN (P35908) Keratin
CLUS_HUMAN (P10909) Clusterin precursor CLUS_HUMAN (P10909) Clusterin precursor
RS3A_MOUSE (P97351) 40S ribosomal protein S3A RS3A_MOUSE (P97351) 40S ribosomal protein S3A
T13B_HUMAN (Q9Y275) Tumor necrosisfactor ligand superfamily member 13 (BAFF)
EF11_HUMAN (P04720) Elongation factor1-alpha 1 (EF-1-alpha-1) (Elonga
EF11_HUMAN (P04720) Elongation factor1-alpha 1 (EF-1-alpha-1) (Elonga
• One major evolution of proteomics technologies in the last years has been the introduction of gel-free approaches for large scale protein identification and quantification
• These methods combine isotope labelling, separation techniques and mass spectrometry
WORKFLOWS 3: WORKFLOWS 3: isotope labelling strategiesisotope labelling strategies
Relative quantification :
Comparison of proteins from samples A vs B ? Which proteins change in amount and how much ?
Applications :
-Healthy vs. diseased tissues
-Healthy vs. diseased body fluids
-Drug treated / untreated cells
-Stimulated / unstimulated cells
-Mutants / wt cells-……..
+TOF MS: Experiment 1, 44.071 to 46.012 min from 181203_QS_MQ_RuedaICAT1_long...a=3.56275471721098790e-004, t0=7.24150134716619500e+001
Max. 649.4 counts.
930 940 950 960 970 980 990 1000 1010 1020m/z, amu
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
Inte
ns
ity, c
ou
nts
8.96
968.52
9.96
1.00
10.962.00
m = 9 Da
Peptide from sample A
Peptide from sample B
Relative quantitation : stable isotope labelling is very fashionable!
Sample A : light isotope Sample B : heavy isotope
mix, digest
Quantitate and identify ( MS)
How to label ?
-chemically, post protein synthesis
“specific” chemical modification of AA side chain
(+) any sample can be done(-) side reactions
-metabolically, during protein synthesis
Incorporation of one or more labelled amino acid(+) “native” proteins(-) need cultivable organism
d0- or d8-ICATd0- or d8-ICAT
Biotin Biotin tagtag
Linker (heavy or Linker (heavy or light)light)
SS
NN NNOO
NN OOOO
OO NNII
OO OOXX
XX
XX
XX
XX
XX
XX
XX
ThiolThiolreactivereactive
(X= H or D)
State 1 State 2Transition states
[protein1][protein2]
.
.[proteinn]
[protein1][protein2]
.
.[proteinn]
Isotope Coded Affinity Tag (ICAT) reagents
Cell State 1 Cell State 2
Combine samples
Inte
nsity
m/z
A1
A2A3B1
B2Identify proteins by MS/MS
Inte
nsity
m/z
aa1aa2aa3
aa4
Quantitate protein levels by H8 / D8 peak heigth ratios
New Methods :New Methods : ICAT:quantitation ICAT:quantitation and identificationand identification
Biotin NH
O IHH
HH HH
HHBiotin N
H
O IDD
DD DD
DD
Modify with (H8)-ICAT
Modify with (d8)-ICAT
•Digest Trypsin•Purify Cys-containing peptides on avidin column
HS- -SH
Avidin Affinity Chromatography
control treatment
ICAT labelICAT label
Combine & proteolyzeCombine & proteolyze
d0d0
d0d0/d8d8
d8d8
Fraction # (Time (min))Fraction # (Time (min))
KC
l K
Cl
[M]
[M]
0.00.00.10.10.20.20.30.30.40.40.50.5
0.00.0
0.30.3
0.60.6
70701010 2020 3030 4040 5050 6060
Ion-Exchange
OD
OD
22 1414
%
%
AcN
AcNR
.A.
R.A
. (%
)(%
)
00
5050
100100
00
100100
5050
Time (min)Time (min)1010 2020 4040 5050 6060 70703030
LC-MS/MS
m/zm/z400400 800800 12001200 16001600 20002000
00
5050
100100 y9 y12y4 y8y5 y11 y14 y15 y17y13 y16y7y6 y10
b15b4 b10 b14b7 b8b6b3b2 b5 b9 b11 b12 b13R.A
. R
.A.
(%)
(%) D SQTNI N I AL T D A
A SLTAD NIQ NT DI
Area = 1.21x109
Area = 1.01x109
d0d0
d8d8 d8d8//d0d0 = 1:0.83
A) Identify
B) Quantify
1)
2)
3)
4)
5)
6)
Pair wise ICAT with Multidimensional Chromatography
ICAT (+) and (-)
- relative protein quantification by MS
- simplification of complex mixtures by selecting a subset of peptides after digestion
- eliminate analytical variability by mixing samples
~15 different isotope labelling methods developed in the last 5 years !!
- protein quantification unreliable for weak signals
- affinity purification (avidin) : losses for low amounts
- multiple side reactions possible
+
-
Recent ICAT studies (R. Aebersold’s group)Wollscheid B, von Haller PD, Yi E, Donohoe S, Vaughn K, Keller A, Nesvizhskii AI, Eng J, Li XJ, Goodlett DR, Aebersold
R, Watts JD.Lipid raft proteins and their identification in T lymphocytes.Subcell Biochem. 2004;37:121-52
Yan W, Lee H, Yi EC, Reiss D, Shannon P, Kwieciszewski BK, Coito C, Li XJ, Keller A, Eng J, Galitski T, Goodlett DR, Aebersold R, Katze MG.
System-based proteomic analysis of the interferon response in human liver cells .Genome Biol. 2004;5(8):R54.
Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A, Wijgers N, Jaspers NG, Raams A, Argentini M, van der Spek PJ, Botta E, Stefanini M, Egly JM, Aebersold R, Hoeijmakers JH, Vermeulen W.
A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A.Nat Genet. 2004 Jul;36(7):714-9.
Ranish JA, Hahn S, Lu Y, Yi EC, Li XJ, Eng J, Aebersold R.Identification of TFB5, a new component of general transcription and DNA repair factor IIH.Nat Genet. 2004 Jul;36(7):707-13.
Hardwidge PR, Rodriguez-Escudero I, Goode D, Donohoe S, Eng J, Goodlett DR, Aebersold R, Finlay BBProteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli.J Biol Chem. 2004 May 7;279(19):20127-36.
Zhang J, Goodlett DR, Peskind ER, Quinn JF, Zhou Y, Wang Q, Pan C, Yi E, Eng J, Aebersold RH, Montine TJ. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid.
Neurobiol Aging. 2005 Feb;26(2):207-27.
Marelli M, Smith JJ, Jung S, Yi E, Nesvizhskii AI, Christmas RH, Saleem RA, Tam YY, Fagarasanu A, Goodlett DR, Aebersold R, Rachubinski RA, Aitchison JD.
Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.
J Cell Biol. 2004 Dec 20;167(6):1099-112. Epub 2004 Dec 13.
• Label light / heavy cultures(Leu d0 / d3)
• Stimulate heavy cells
• Mix cells or lysates
• Purify fraction of interest
• Analyse by LC-MS/MS (->ID)
• Quantify signals of ion pairs
SILAC
Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M.
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
Mol Cell Proteomics. 2002 May;1(5):376-86.
SILAC (+) and (-)
• relative protein quantification by MS
• eliminate praparative variability by mixing samples immediately after culture
• eliminate analytical variability
• peptides in native state (no side reactions)
• protein quantification unreliable for very weak signals
• mass shift variable (dependent on number of residues)
• only feasible with organisms in culture
+
-
Recent SILAC articles
Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M.Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol Cell Proteomics. 2002 May;1(5):376-86.
Blagoev B, Ong SE, Kratchmarova I, Mann M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.Nat Biotechnol. 2004 Sep;22(9):1139-45. Epub 2004 Aug 15.
Gruhler A, Olsen JV, Mohammed S, Mortensen P, Faergeman NJ, Mann M, Jensen ON. Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway.Mol Cell Proteomics. 2005 Mar;4(3):310-327.
de Hoog CL, Foster LJ, Mann M.RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers.Cell. 2004 May 28;117(5):649-62.
Ong SE, Kratchmarova I, Mann M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC).J Proteome Res. 2003 Mar-Apr;2(2):173-81.
Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M.A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat Biotechnol. 2003 Mar;21(3):315-8. Epub 2003 Feb 10.
Foster LJ, De Hoog CL, Mann M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors.Proc Natl Acad Sci U S A. 2003 May 13;100(10):5813-8. Epub 2003 Apr 30.
Other fields
• Proteome subsets– Phosphoproteome – Ubiquitinated proteins– …
• Clinical proteomics (marker discovery)– Too vast to summarise
• Proteome imaging– MALDI of tissues