Transcript PAF_PROTEOMICS_INTRO

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 :
Research :
•Teaching :
•Identification of gelseparated proteins by MS
•Development of
proteomics methods
•Techniques, possibilities
& limitations of
proteomics approaches
•Project discussion
•Applications to
biological problems
•2D-PAGE and/or other
multi-dimensional
separation techniques
PAF technology and instrumentation
260404RPChistonesFRAC001:1_UV1_280nm
260404RPChistonesFRAC001:1_Inject
260404RPChistonesFRAC001:1_Conc
260404RPChistonesFRAC001:1_Fractions
mAU
30.0
20.0
10.0
0.0
-10.0
-20.0
-30.0
A1
0.0
1D-electrophoresis
2D-electrophoresis
Nano-HPLC- Quadrupole-time-of-flight
Mass spectrometer
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
5.0
10.0
15.0
20.0
25.0
Liquid Chromatography
Nano-HPLC-triple quadrupole ion trap
Mass spectrometer
ml
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,…
Overview : classes of proteomics experiments
Protein expression
analysis
Interaction / Functional
Proteomics
FOCUS :
Complex samples
Whole proteomes
200 and more proteins
FOCUS :
Subcellular fraction
Organelle
Protein Complex
20-200 proteins
FOCUS :
Single protein
PTM analysis
1-20 proteins
Analytical
Detail
Sample
complexity
Proteomics
• 2D-PAGE
• Mass Spectrometry for proteomics
• Proteomics workflows and applications
2D-PAGE
IEF: the principle
How to create a pH gradient ?
Improvements in 2D-PAGE
+
-
pH 3.0
10.0
- IPG (Immobilised Ph Gradient) strips for the first dimension
pH-forming chemical groups are grafted onto the
polyacrylamide matrix, creating a mechanically stable pH gradient
++
++
++
++
mechanical stability
reproducibility
loadable amounts
„zoom“ pI ranges
After IEF : equilibration and 2nd dimension
+
-
pI
Equilibration step 1 :
•SDS
•Buffer pH 6.8
•DTT (reduce –S-S-)
Equilibration step 2 :
•SDS
•Buffer pH 6.8
•Iodoacetamide
(alkylate –S-S-)
3.0
5.5
6.5
8.5
10
150
100
75
50
37
20
10
One protein  many spots
Post-translational
modifications can result in
changes in pI and/or MW
Spot “trains” for extensively
modified proteins
Caused by
Glycosylation
Phosphorylation
Acetylation (K)
…
Database of 2D images
with clickable spots :
www.expasy.org/ch2d/
2D-PAGE and image analysis are used for
studying changes in composition of the
proteome
Control
Stimulus applied
Software-based image analysis
•Spot detection
•Spot quantification
•Gel-to-gel
•Matching
•Presence/absence
of spot
•Up/down regulation
•Statistic analysis
Mass Spectrometry in proteomics
Mass spectrometry : essential functions
SAMPLE
ION SOURCE
MASS ANALYZER
ION
GENERATION
ION
SEPARATION
ESI : Electrospray Ionisation
Quadrupoles
Ion traps
Time-of-flight with reflectron
TOF/TOF
FT-ICR
(Fourier transform –
Ion Cyclotron Resonance)
MALDI : Matrix Assisted Laser
Disorption/Ionization
DETECTOR
ION
DETECTION
Faraday cup
Scintillation counter
Electromultiplier
High-energy dynodes with
electronmultiplier
Array (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 IONISATION
MALDI (Matrix Assisted
Laser Desoprtion Ionisation
MALDI TOF (Time Of Flight)
MALDI TOF
• 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
disadvantages
•High accuracy needs careful calibration
•Difficult (but possible) to do MS/MS by MALDI
•Signal suppression in complex mixtures
•Crystallisation conditions influence results
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
927.59
Max. 1305.0 counts.
YLYEIAR
190
180
170
160
150
In te n s ity , c o u n ts
140
LGEYGFQNALIVR
130
120
HPEYAVSVLLR
110
LSQKFPK
100
847.59
LVNELTEFAK
HLVDEPQNLIK
1440.00
80
FKDLGEEHFK
?
70
?
60
869.07
40
1249.77
?
30
871.07
857.14
10
800
900
1024.56
1050.55
978.60
1000
1567.94
1640.16
1163.77
20 789.53
1479.98
1022.56
50
0
DAFLGSFLYEYSR
?
90
1073.03
1108.71
1200
1481.98
1296.86
1283.91
1142.86
1100
1305.87
KVPQVSTPTLVEVSR
1292.95
1300
m/z, amu
?
1417.93
1443.01
1386.76
1501.84
1400
1500
?
1595.95
1824.09
1616.92 1790.10
1600
1700
1800
ELECTROSPRAY IONISATION
• molecules compete
for ionisation
e.g. Na+ >> Peptide
P. Kebarle, M. Peschke / Analytica Chimica Acta 406 (2000) 11–35
ELECTROSPRAY IONISATION
• Great for MS/MS
– Can be directly coupled to reversed phase LC
(separation !)
– Sensitive
– Excellent for MS/MS due to 2+/3+ ions
disadvantages
•Sample introduction more complex
•Data analysis more difficult (2+/3+ ions)
•one-shot sample analysis (time constraints)
•Very low tolerance to contaminants
1+ versus 2+ ions
1163.76
84
582.28
200
80
75
180
70
Dm=1.0 Da
65
Dm=0.5 Da
160
60
140
55
582.77
1164.76
Intensity, counts
Intensity, counts
50
45
40
35
30
1.0 Da
25
120
100
80
0.5 Da
60
583.27
20
1165.74
1.0 Da
40
15
0.5 Da
1166.78
10
1162.70
20
5
0
1160
1162
1164
1166
1168
m/z, amu
1+
1170
1172
1174
0
580.0
581.0
582.0
583.0
584.0
m/z, amu
2+
585.0
586.0
Modes of measurement : MS & MS/MS
•Ion production (ionisation)
•Ion separation
MS
•Ion detection
•Ion production (ionisation)
•Ion separation – isolation of “parent” ion
•Ion fragmentation (CID)
•Ion separation – separate fragment ions
•Ion detection - measure fragment ions
Tandem MS
MS/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 thedissociation
acquisition of multiple orthogonal data files (1
•CID=
collision
induced
spectrum / peptide)
•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
MS/MS Glossary and facts
•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.
Covalent bonds being broken  ion series
Covalent bonds being broken  ion series
ESI & Quadrupole-based instruments
Triple Quadrupole
Triple QuadrupoleIon trap
Quadrupole-Quadrupole
TOF
Ion Trap (3D trap)
All these instruments can perform MS/MS fragmentation experiments
• Identifying proteins by mass spectrometry
MS-based protein identification : general
concept
experimental
In silico
Protein sample
Protein sequence(s)
Specific protease
e.g. trypsin
software
Protein fragment sequences
(same protease specificity)
Protein fragments
(5-30 AA peptides)
MS
software
Exact masses of peptides
Calculated exact masses of peptides
software
Fragmentation (MS/MS)
spectrum of each peptide
Calculated fragmentation spectrum
of each peptide
Best Match(es)
• Protein identification by Peptide Mass
Fingerprinting (PMF)
Information contained in MS spectrum
Extracted peak list
m/z
+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.
927.59
180
170
160
150
140
In te n s ity , c o u n ts
847.5896
869.0722
922.5712
923.5815
927.5904
1022.5551
1050.5533
1163.7695
1164.7531
1193.7393
1249.7705
1250.8103
1296.8556
1297.8499
1305.8668
1416.8929
1440.0008
1479.9773
1482.9583
1567.9417
1640.1635
1824.06
…
190
130
120
110
847.59
100
90
1440.00
80
1479.98 1567.94
70
869.07
60
1640.16
1022.56
50
1163.77
40
1249.77
30
20 789.53
857.14
10
0
871.07
800
900
1024.56
1050.55
978.60
1000
1073.03
1481.98
1296.86
1283.91
1142.86
1108.71
1100
1305.87
1200
1417.93
1386.76 1443.01 1595.95
1824.09
1292.95
1501.84
1616.92 1790.10
1300
1400
m/z, amu
1500
1600
1700
1800
Search form…
Results page…
• Protein identification by MS/MS
Experimental set-up : nanoLC-MS/MS
HPLC
pumps
~95%
further analysis
(waste)
~5
%
T-splitter
sample
Mass spectrometer
C18 Column
L = 10 cm
ID = 50-100 µm
database correlation
An on-line LC-ESI-MS experiment with automatic data acquisition
MeCN
gradient
568.58
3.6e4
496.57
3.2e4
354.11
563.23
445.07
2.8e4
461.71
Mr peptide : 921.4
Intensity, cps
2.4e4
2.0e4
1.6e4
1.2e4
8000.0
4000.0
0.0
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Time, min
+TOF Product (461.7)
20
86.09
K
147.01 I
Intensity, counts
15
D
F
L
y2
y1
175.06
I
S
S
y5
635.31
260.17
y4
270.11
10
522.23
y3
y6
375.19
5
748.39
359.01
y7
0 50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
y8
900
m/z, amu
Automated LC- MS/MS run
Chromatogram :
Total ion current vs. time
421.68
1.00e5
653.32
464.17
722.28
582.25
501.73
507.75
5.00e4
0.00
25
30
35
1
Full scan : +TOF MS:
40
45
50 Time, min55
595.29
740.41
65
70
60
700.34
75
80
1553 (3+)
518.82
400
2
4
200
3
507.1541
0
400
450
MS/MS peptide 4 :
+TOF Product (662.8):
5.0
0.0
500
1553 (2+)
662.76
550
600
650
777.8176
700
750
800
850
900
m/z, amu
235.08
14.0
10.0
531.47
536.80
166.06
147.10
120.06
110.04
138.06
100
262.10
207.09
409.18
614.2136
200
300
400
500
600
663.3252
700
800
900
1000
1100
1200
m/z, amu
1300
Matching of MS / MS data
b2
251.12
+TOF Product (653.3)
19.7
19.0
Residue
Immonium a
b
y
----------------------------------------------------------------------------------H, His
110.07
110.07
138.06
1305.71
L, Leu
86.09
223.15
251.15
1168.65
V, Val
72.08
322.22
350.21
1055.57
D, Asp
88.03
437.25
465.24
956.50
E, Glu
102.05
566.29
594.28
841.47
P, Pro
70.06
663.34
691.34
712.43
Q, Gln
101.07
791.40
819.39
615.38
N, Asn
87.05
905.44
933.44
487.32
L, Leu
86.09
1018.53
1046.52
373.28
I, Ile
86.09
1131.6157 1159.61
260.19
K, Lys
101.10
1259.7106 1287.70
147.11
18.0
17.0
16.0
15.0
a1
14.0
I
110.06
13.0
a2
223.13
12.0
Intensity, counts
11.0
10.0
9.0
MH22+
precursor
8.0
b1
7.0
138.05
y1
653.36
6.0
332.21
5.0
b5
I
4.0 86.09
I
3.0
2.0
1.0
594.25
b3
y6
y9
350.24
712.46
a5
1055.49
y3 b4
y7
y8
y10
373.30465.16566.32
166.05
841.53 956.41
1168.56
b8
206.16
y2
0.0
100
200
300
400
500
600 700 800
m/z, amu
900 1000 1100 1200 1300
Black : predicted
Red : predicted and detected
Mascot 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
19
31
33
47
58
60
61
64
74
97
98
461.80
501.80
569.80
582.30
653.40
722.40
739.80
740.40
751.90
547.30
627.70
636.70
921.58
1001.58
1137.58
1162.58
1304.78
1442.78
1477.58
1478.78
1501.78
1638.88
1880.08
1907.08
921.48
0.10 0
1001.58 0.01 0
1137.49 0.09 0
1162.62 -0.04 0
1304.71 0.08 0
1442.63 0.15 0
1477.52 0.07 0
1478.79 -0.00 0
1501.61 0.18 0
1638.93 -0.05 1
1879.91 0.16 0
1906.91 0.16 0
55
31
72
76
90
87
43
61
37
85
34
42
1
1
1
1
1
1
1
1
1
1
1
1
AEFVEVTK
LVVSTQTALA
CCTESLVNR
LVNELTEFAK
HLVDEPQNLIK
YICDNQDTISSK
ETYGDMADCCEK
LGEYGFQNALIVR
EYEATLEECCAK
KVPQVSTPTLVEVSR
RPCFSALTPDETYVPK
LFTFHADICTLPDTEK
Orthogonal datasets and confidence levels
PMF :
MS/MS :
one MS spectrum
 one dataset (peak list)
n MS/MS spectra
 n orthogonal datasets
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
Summary : Typical Analytical Workflow
Biological
question
Chromatographic
Separation (reversed-phase)
Protease
digestion
Peptide
extraction
Nano-HPLC
time
m/z
Tandem mass
spectra of 502000 peptides
MS/MS
Output :
Database searching
Software (MASCOT)
Protein sequence
database
•Protein identification in
simple/complex mixtures
•Extensive sequence
coverage and peptide
mapping
•Analysis of modified
peptides possible
Database matches
DHX9_HUMAN
ATP-dependent RNA helicase A
NFM_HUMAN
Neurofilament triplet M protein
Q9BQG0
Hypothetical protein
MYO6_HUMAN
Myosin VI.
TP2A_PIG
DNA topoisomerase II, alpha isozyme
Q7Z5Y2
Rho-interacting protein 3.
FLIH_HUMAN
Flightless-I protein homolog.
TP2B_MOUSE
DNA topoisomerase II, beta isozyme
S3B1_HUMAN
Splicing factor 3B subunit
Q8VCW5
Similar to alpha internexin neuronal
Q8CHF9
MKIAA0376 protein (Fragment).
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
FSLCILTPEK
1369.75
24
LSTHELTSLLEK
1406.77
55
FFILYEHGLLR
1775.88
16
QVPIAPVHLSSEDGGDR
Caveat : Protein identification
IS NOT
protein characterisation
Two peptides are enough to identify a protein
But
We are still identifying two peptides, not the entire protein
Highly similar sequences cannot be
distinguished
For finding PTMs extensive
Sequence coverage is essential !!
2
Technology, workflows and
applications : what is available
New and old tools
Genome sequence databases
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
Protein sequence databases
Biological knowledgebases :
- functions
- pathways
- seq. motifs
- 3D structures
WORKFLOWS 1 :
„classical“
2D-PAGE
+
MALDI TOF
Workflow 1 : adaptation of bacteria to growth conditions
Normal medium
1
Low Glucose
2
3A 3B
4
8 7
9
12
2
1
3A 3B
8 7
56
9
10
11
12
15
8
14
4
6
10
11
E.Coli adapts to a very low
glucose medium by
up- and downregulating
a set of 15 proteins
Wick LM, et al, Environ Microbiol 3: 588-599, 2001
Workflow 1 : adaptation of bacteria to growth conditions
M5
M3
M2
M1
M8
M4
M7
M6
Tryptic digestion
Peptide Mass
Fingerprinting
Search with mass list
M1......M8
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
Metabolic
enzymes and
transport
proteins affected
Utilisation of
alternative
sources of energy
WORKFLOW 1 : quantitation and kinetics
AldA (1)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
0.12
0.10
0.08
0.06
0.04
0.02
0
14
12
10
8
6
4
2
0
3.0
2.5
2.0
1.5
1.0
0.5
0
1 2 3 4 5
UgpB (4)
1 2 3 4 5
MalE (8)
1 2 3 4 5
ArgT (12)
1 2 3 4 5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
18
16
14
12
10
8
6
4
2
0
0.25
0.20
0.15
0.10
0.05
0
DppA (2)
1 2 3 4 5
AtpA (5)
1 2 3 4 5
MglB (9)
1 2 3 4 5
6
5
4
3
2
1
0
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
AceA (3A)
1 2 3 4 5
YdcS (6)
1 2 3 4 5
3.0 GatY (10)
2.5
2.0
1.5
1.0
0.5
0
1 2 3 4 5
1.2
1.0
0.8
0.6
0.4
0.2
0
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
AceA (3B)
1 2 3 4 5
LivJ (7)
1 2 3 4 5
RbsB (11)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1 2 3 4 5
Time points :
1) Start – batch
culture
2) Adapted
bacteria put
back
into highglucose batch
culture
3) 40 hr
adaptation
culture
4) 156 hr
adaptation
culture
5) 500 hr
adaptation
culture
MalI (13)
Relative protein quantities
as a function of time during
the adaptation process
1 2 3 4 5
Why is SDS-PAGE such a good preparation method?
•
•
•
•
•
•
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
Disadvantages
• protein digestion in gel: non quantitative
• peptide sequence recovery: usually incomplete
• whole protein recovery: poor
In-gel digestion: solid phase chemistry of proteins
WORKFLOWS 2 :
General shotgun protein identification
techniques
example :
Affinity pull-down
+
1D-PAGE
+
LC-MS/MS
Shotgun sequencing from complex mixtures
Denaturation,
Proteolytic
digestion
Multiprotein complex
Complex peptide mixture
(1000-20000 species)
List of identified proteins
1.
2.
3.
4.
5.
6.
P45218
P21543
Q12588
P32651
Q01245
….
Rp-LC-MSMS run
Db search
Alternative : MuDPIT (Multi Dimensional Protein Identification
Technology)
Post-digestion separation of peptides by two-dimensional liquid chromatography
instead of separation of proteins
Strong
Cation
Exchange
(SCX)
separation
Multiprotein mixture
(complex)
Denaturation,
Proteolytic
digestion
Complex peptide
mixture
(1000-20000 species)
List of identified proteins
1.
2.
3.
4.
5.
6.
P45218
P21543
Q12588
P32651
Q01245
….
Db search
Rp-LC-MSMS runs
A VERY complex mixture – direct analysis (no separation)
TIC: from 151002_ACO_B4strep.wiff
Max. 5.3e5 cps.
653.36
5.3e5
133.06
5.0e5
526.26
4.5e5
406.23
303.13
In te n s ity , c p s
4.0e5
3.5e5
3.0e5
852.44
199.19
402.54
203.11
186.12
472.25
541.11
86.10
536.34
574.66
1064.42
449.15
629.30
494.25
665.38
2.5e5
330.18
2.0e5
527.26
615.41
86.10
563.30
1.5e5
599.32
1.0e5
409.18
5.0e4
0.0
5
10
15
20
25
30
35
40
45
50
55 60 65
Time, min
70
75
80
85
90
95
100 105 110 115
A VERY complex mixture still gives results, but...
Mascot Search Results
User : 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
. . .
........ how deep are we going ?
Intensity, cps
2.7e5
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
120.08
157.15
545.75
545.75
581.28
652.36
216.11
670.84
131.09
343.18
738.39
555.22
153.08
561.29
548.74
681.35
498.55
569.27
489.53
445.07
499.72
499.70 469.25
469.24
651.85
445.10
18
20
22
24
26
28
30
32
34
36
38
40
42
445.09 648.38
44
46
48
Time, min
180
analyzed
missed
160
Intensity, counts
140
120
100
80
60
40
20
10
0
450
500
 some protein
prefractionation
550
600
650
is necessary
! 700
750
m/z, amu
800
850
900
TNF family of ligands and TNF-receptor family
Bodmer JL et al, TIBS. 2002 27(1):19-26
Analysis of apoptotic signalling complexes
The Fas (CD95) signalling complex (DISC)
(-)
FasL
(+)
FasL
?
Fc-FasL
Casp.10
Casp.8
Flip
?
Fas
FADD
Model
Western blot
Real life
Analysis of apoptotic signalling complexes : negative control
(-)
BAFF
(+)
FasL
(-)
BAFF
(+)
FasL
1 cm
* run 1 cm
* no fix, no stain !
* cut, digest
LC-MS/MS
ANALYSIS
FasL HL
search results
BAFF HL search results
Database
: MSDB 200402 (851746 sequences; 265326103 residues)
: MSDB
200402
(851746
sequences; 265326103 residues)
TaxonomyDatabase
: Mammalia
(mammals)
(166849
sequences)
Taxonomy
: Mammalia (mammals) (166849 sequences)
Significant hits:
Significant
hits:
A37241
52K autoantigen Ro/SS-A - human
TIC: from 180702BaffHL.wiff
BAFF complex
HL sample
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
2.8e5
In te n s ity , c p s
2.4e5
2.2e5
2.0e5
LCMS runs Fas/Baff
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
5
10
15
20
25
30
TIC: from FAS_180702_HL.wiff
4.0e5
3.8e5
FasL complex
HL sample
3.6e5
3.4e5
3.2e5
3.0e5
2.8e5
In te n s ity , c p s
Q8WUC1 Q96RF8
TUBULIN,
SSA1.BETAHomo
5.- Homo
sapiens
sapiens
(Human).
(Human).
C25437 BAB27292
tubulin beta-3
AK010960
chain - mouse
NID: - Mus musculus
CAC39526K2C1_HUMAN
SEQUENCE Keratin,
15 FROMtype
PATENT
II cytoskeletal
WO0129232.-HUman
1 (Cytokeratin 1)
AAH19046A45935
SIMILARdnaK-type
TO IMMUNOGLOBULIN
molecular chaperone
HEAVY hsc70
CONSTANT
- mouse
GAMMA 3
K1CJ_HUMAN
KRHU0 Keratin,
keratin
type 10,
I cytoskeletal
type I, cytoskeletal
10 (Cytokeratin
- human10-(Human).
I37383 C25437
FAS soluble
tubulin
protein
beta-3
- human
chain - mouse
A24903 Q9CWA2
tubulin alpha-1
ATP chain
SYNTHASE,
- Chinese
H+ hamster
TRANSPORTING MITOCHONDRIAL F1
A44861 CAA30026
keratin, 67KHSHA44G
type II epidermal
NID: - Homo
- human
sapiens
I38707 CAB59134
Fas ligand - human
SEQUENCE 1 FROM PATENT WO9818921 PRECURSOR
AAG41947A44861
AF304164
keratin,
NID: 67K
- Homo
typesapiens
II epidermal - human
Q9BDN1 Q9BWB7
CD95L PROTEIN.HEAT SHOCK
Cercocebus
70KD PROTEIN
torquatus9B
atys
(MORTALIN-) (Human).
CAA82315A26168
HSKERAT9
ribophorin
NID: - Homo
I precursor
sapiens
- human
AAB86467PT0207
IMMUNOGLOBULIN
Ig gamma chain
GAMMA
C region
HEAVY
- chimpanzee
CHAIN (Human).
NUCL_HUMAN
PWHUA Nucleolin
H+-transporting
(Protein C23).two-sector
(Human).ATPase alpha chain precursor - human
1ATS
JC1473
heat shockH+-transporting
cognate proteinATP
70 kD
synthase
(44 kD chaperone
(EC 3.6.1.34)
ATPase
alpha chain - mouse
1FC1A I77403
Ig gamma-1
tubulin
chain
alpha-1
C region
chain
(Fc-fragment),
human
chain A - human
I61769 AAA57233
keratin 6d, type
MUSHP7A2
II - human
NID:
(fragment)
- Mus musculus
A29904 CAA82315
keratin 5, type
HSKERAT9
II, epidermal
NID: - -human
Homo sapiens
A40389 A29904
translation
keratin
elongation
5, type
factor
II, epidermal
eEF-1 alpha
- human
chain (clone pS1) - rat
HHHU84
HSU86214
NID:
shock
- Homo
protein
90-beta
- human
35
40
45
50
55 AAB46730
60
65
70
75 heat
80
85
90sapiens
95
100[validated]
105
110
115
Time,
min AAB86467
CAD23746
IMMUNOGLOBULIN
IMMUNOGLOBULIN
GAMMA HEAVY
GAMMA
CHAIN
HEAVY
CONSTANT
CHAIN .-(Human).
REGION
Q10466 B26168
TITIN, HEART
ribophorin
ISOFORM
II precursor
N2-B (EC- human
2.7.1.-) (CONNECTIN).-(Human).
Q8WZ42 CAA34756
TITIN.- Homo
HSEF1AC
sapiensNID:
(Human).
- Homo sapiens Max. 4.0e5 cps.
1D3OA S21097
trypsin (EC
alpha-1-antitrypsin
3.4.21.4), chain Aprecursor
- pig
- bovine
603.33
CAC20457S04652
IMMUNOGLOBULIN
Ca2+-transporting
HEAVY
ATPase
CHAIN(EC
CONSTANT
3.6.1.38) GAMMA
2, - pig 4.- (Human).
CAA41735
HEAT SHOCK
BTBSA
PROTEIN
NID: 70
- Bos
TESTIS
taurus
VARIANT.- (Human).
655.82 O75634
Q8WTZ6 Q9UK02
RIBOSOMAL
BIP PROTEIN (FRAGMENT).L18.- Homo sapiens
Homo(Human).
sapiens (Human).
1NBMC KRHUEA
f1-atpase (EC
keratin
3.6.1.34)
6a, type
delta
II -and
human
1 epsilon subunits, chain C - bovine
Q96PE2 A22224
TUMOR ENDOTHELIAL
actin alpha, vascular
MARKER
smooth
4.- Homo
muscle
sapiens
- mouse
(Human).
Q9R1Q3
Q96GA6
GLIAL
FIBRILLARY
UNKNOWN
ACIDIC
(PROTEIN
PROTEIN
FOR
MGC:15420).ALPHA.Rattus
Homo
norvegicus
sapiens (Rat).
(Human).
328.19
ITSH
Q8WZ42
alpha-1-antitrypsin
TITIN.- Homo
precursor
sapiens
- sheep
(Human).
130.11
CAA27396I84741
MMACTBR2
RNA helicase
NID: - Mus
- mouse
musculus
Q8WXH0
Q9TS10
NUANCE.78Homo
KDA APAMIN
sapiens BINDING
(Human).PROTEIN.- Bos taurus (Bovine).
405.33
Q9GL40 AAH02690
FAS ANTIGEN.BC002690
Macaca
NID:
mulatta
- Homo
(Rhesus
sapiens
macaque).
337.17
A33370
I48385
H+-transporting
RNA
helicase
ATP
synthase
TNZ2
mouse
beta
chain
precursor, mitochondrial
864.45
CAB76567Q96FZ6
MMU250841
HEAT
NID:
SHOCK
- Mus
60KD
musculus
PROTEIN 1 (CHAPERONIN).(Human).
171.17
582.29
Q9GK28 AAA56753
FAS ANTIGEN
HSU15637
APO-1/CD95.NID: - Homo
Macaca
sapiens
arctoides (Stump-tailed macaque).
Q9BZL4 CAA58470
MYOSIN BINDING
HSPXMP11
SUBUNIT
NID: -85.Homo
Homo
sapiens
sapiens (Human).
643.87
603.69
…………………………………
JQ0028
cytokeratin
19 – mouse
…………………………………
158.06
600 spectra
2.6e5
0.0
Max. 3.8e5 cps.
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
680 spectra
186.16
6.0e4
171.18
120.11
620.67
607.38
449.15
4.0e4
2.0e4
0.0
5
10
15
20
25
30
35
40
45
50
55
60
65
Time, min
70
75
80
85
90
95
100
105
110
115
FILTERED RESULTS
Database
search
1.
2.
3.
4.
1.
2.
3.
4.
1.
2.
3.
4.
Database
search
Database
search
Total list sample 1
1.
2.
3.
4.
5.
6.
7.
Protein 1
Protein 2
Protein 3
Protein 4
Protein 5
Protein 6
…
Protein 1
Protein 2
Protein 3
…
Protein 1
Protein 2
Protein 3
…
Protein 1
Protein 2
Protein 3
…
Total list sample 1
1.
2.
3.
4.
5.
6.
7.
Protein 1
Protein 2
Protein 3
Protein 4
Protein 5
Protein 6
…
Total list sample 2
1.
2.
3.
4.
5.
6.
7.
Protein 1
Protein 2
Protein 3
Protein 4
Protein 5
Protein 6
…
Subtract from each list :
1)
COMMON CONTAMINANTS ( PROTEINS STICKING TO BEADS)
2)
LIGAND-COPURIFYING PROTEINS (EX SJOGREN SYNDROME 52 KDA)
3)
COMMON HITS (WHAT IS IN BOTH LISTS)
FILTERED RESULTS
MASCOT DATABASE SEARCH
Fas (CD95) complex
ICE8_HUMAN
MASCOT DATABASE SEARCH
BAFF receptor complex
(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
regulator
(O15519) CASP8 and FADD-like apoptosis
RS8_HUMAN
(P09058) 40S ribosomal protein S8
GBLP_HUMAN
protein
(P25388) Guanine nucleotide-binding
GBLP_HUMAN
protein
(P25388) Guanine nucleotide-binding
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 necrosis
factor ligand superfamily member 13 (BAFF)
EF11_HUMAN
(P04720) Elongation factor
1-alpha 1 (EF-1-alpha-1) (Elonga
EF11_HUMAN
(P04720) Elongation factor
1-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:
isotope 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
-……..
Relative quantitation : stable isotope labelling is very
fashionable!
Sample A : light isotope
Sample B : heavy isotope
mix, digest
Quantitate and identify ( MS)
+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.
8.96
320
300
Dm = 9 Da
280
260
Peptide from sample B
Intensity, counts
240
220
200
Peptide from sample A
180
160
968.52
140
9.96
120
100
80
1.00
60
40
10.96
2.00
20
0
930
940
950
960
970
m/z, amu
980
990
1000
1010
1020
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
Isotope Coded Affinity Tag (ICAT) reagents
Transition states
State 1
State 2
[protein1]
[protein2]
.
.
[proteinn]
[protein1]
[protein2]
.
.
[proteinn]
O
N
N
XX
O
N
S
Biotin
tag
XX
O
O
XX
O
O
XX N
Linker (heavy or
light)
I
Thiol
reactive
d0- or d8-ICAT
(X= H or D)
Cell State 1
New Methods :
ICAT:quantitation
and identification
Modify with
(H8)-ICAT
HH HH
Biotin
HH HH
O
N
H
Cell State 2
Modify with
(d8)-ICAT
DD DD
I
O
Biotin
Combine samples
N
H
DD DD
HS-
-SH
•Digest Trypsin
•Purify Cys-containing
peptides on avidin column
Intensity
aa4
aa3
aa2
Intensity
Identify proteins by MS/MS
aa1
B2
A1
B1
A2
A3
m/z
m/z
I
Quantitate protein levels
by H8 / D8 peak heigth ratios
Pair wise ICAT with Multidimensional Chromatography
treatment
control
1)
A) Identify
2)
d0
R.A. (%)
100
d8
ICAT label
d0/d8
OD214
0.5
0.4
0.3
0.2
0.1
0.0
0.3
30
40
50
60
N
T
b4 b5
A D A T
b6
I
y11 y12
Q S
b7
b8
y14 y15
y16 y17
L T A D A
b9
Q I
L S
y13
b10
b11
T
b12
N
N
b13
b14 b15
I D
800
1200
1600
B) Quantify
Area = 1.21x109
100
Area = 1.01x109
50
0
0
30
N
y9 y10
y8
d0
50
20
50
70
LC-MS/MS
10
I
y7
b2 b3
6)
40
Time (min)
50
60
70
% AcN
R.A. (%)
100
y6
m/z
Fraction # (Time (min))
4)
5)
20
D
400
0.0
10
y5
0
0.6
KCl [M]
3)
Combine & proteolyze
Ion-Exchange
y4
d8
d8/d0 = 1:0.83
2000
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
- protein quantification unreliable for weak signals
-
- affinity purification (avidin) : losses for low amounts
- multiple side reactions possible
~15 different isotope labelling methods developed in the last 5 years !!
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 BB
Proteomic 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.
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.
• 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 (+) 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