Transcript We use 2 types of MS for PROTEIN IDENTIFICATION

My contact details and information about submitting samples for MS
http://www.nottingham.ac.uk/Biosciences/People/susan.liddell
2 main types of MS are used for
PROTEIN IDENTIFICATION
• PEPTIDE MASS FINGERPRINTING (PMF)
– MALDI-ToF MS
• TANDEM MS (aka MSMS)
– ELECTROSPRAY Q-ToF2
Protein digestion
Proteins are chains of amino acids
each of which have slightly different masses
The protein chain can be cut selectively by
sequence specific proteases at particular amino acids
Trypsin cuts after lysine or arginine
The peptides produced have distinct weights
These are accurately measured by mass spectrometry
A list of these weights is like a
fingerprint
(a PEPTIDE MASS FINGERPRINT)
= 89.3
89.3
105.3
89.3
95.4 112.1
= 402.2
95.4
105.3
101.8
97.1
112.1
89.3
= 601
This is unique to the protein
and can be used to identify it
MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionisation)
Laser
energy
Peptide ions enter the
Time of Flight tube
separated on basis
of mass
Peptides cocrystallised with
matrix
Ionise peptides
Detector
mass/charge of
every peptide
peptide mass
fingerprint
peptide mass spectrum
a trypsin digest of a single protein
every peak corresponds to the mass (m/z) of a peptide ion
relative
intensity
m / z = mass / charge
Peptide mass spectrum
a peak list (pkl)
.
.
.
.
Data converted to text
List of peptide masses
=
1051.54
1094.56
1244.64
1476.67
1542.84
1613.88
1664.97
1763.79
1952.89
2264.89
2238.23
.
.
.
.
fingerprint
Database searches with PeptideMassFingerprint data
run digested
protein on MS
sequence
databases
theoretical trypsin
digest of every
predicted protein
list of measured
peptide masses
“fingerprint”
.
.
.
1051.54
1094.56
1244.64
1476.67
1542.84
1613.88
1664.97
1763.79
1952.89
2264.89
2238.23
.
.
.
compare
identification
made if match
is found
list of calculated
peptide masses
peptide mass fingerprinting
• rapid
• high throughput
• large scale identification of proteins from organisms with
completely sequenced genomes
• good tool for a first look at a sample
BUT…….
peptide mass fingerprinting will not always
give an identification
• genome is not completely sequenced
• the full length protein sequence is not in the database
• modifications are present
• more than one protein is present in the sample
alternative method of analysis – LC-MSMS
ElectroSpray Ionisation (ESI)
Mass Spectrometry on the Q-ToF2
Samples in solution
Compatible with HPLC
Complex protein mixtures
Determine peptide masses
Peptide fragmentation
Peptide sequence
MS on the Q-ToF2
MS1
MS2
Tandem MS - peptide fragmentation
low energy collision fragments the peptide
one fragmentation per peptide molecule
cleavage usually occurs at the amide bond i.e. between residues
series of peptide fragments
each fragment is one amino acid longer than the next
the series of fragments corresponds to the sequence of the peptide
peptide fragmentation
strong, approx. 80 kDa 1D gel band
GUY_1_18APRIL07_MSMS_791 MaxEnt 3 29 [Ev-308483,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp)
1: TOF MSMS 791.49ES+
1028.65
y10
100
1563.98
1156.73
y11
1340.85
y13
%
313.20
242.16 b3
b2
757.50
y7
642.47
y6
214.16
a2
0
1269.81
y12
870.58
y8 941.63
y9
426.28
409.25 b4
1139.69
782.44
537.32
147.12
86.10 y1
L
554.34
b5
677.37
791.43
906.45
1010.64
1138.74
1029.75
1191.67
1100
1200
1546.93
1341.74
M/z
100
200
300
400
500
600
700
800
900
1000
1300
1400
1500
the series of fragments corresponds to the sequence of the peptide
On-line LC-MSMS on the Q-ToF2 : peptides from a single protein
Fragment Mass spectrum (MSMS)
fragments from one peptide
Survey Mass Spectrum (MS) - intact peptides
detected in a 1 second survey scan
582.28
100
1F_MP0709_05MAY09 190 (39.732) Cm (190:216)
100
3: TOF MSMS 788.86ES+
1.58e3
MSMS
129.10
1123.53
%
1335.59
242.19
668.31
562.26
789.40
294.18
454.27
913.42
951.45
1238.56
1026.48
732.35
0
m/z
%
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
1163.59
1F_MP0709_05MAY09 169 (37.007) Cm (167:196)
4: TOF MSMS 777.82ES+
1.28e3
662.81
100
MSMS
Many peptides are fragmented during a 60 minute run
235.12
1090.52
LC-MSMS generates much more data than fingerprinting
mass of intact peptides & the fragment masses
Search databases with much more data per protein
%
708.40
837.45
738.85
476.25
409.22
810.74
262.14
147.12
1050.55
1019.48
614.27
778.33
465.17
536.22
207.13
306.16
0
400
m/z
450
500
550
600
650
700
1F_MP0709_05MAY09 194 (37.239) Cm (191:217)
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
MSMS
1F_MP0709_05MAY09 272 (45.212) Cm (271:290)
900.51
2: TOF MSMS 582.28ES+
3.92e3
185.17
100
MSMS
1088.59
1187.67
%
325.19
1412.79
706.89
361.22
1001.57
820.97
490.26
1315.76
589.34
262.16
%
129.11
951.48
0
m/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
595.31
365.22
213.16
708.41
494.27
837.46
147.12
569.27
440.23
326.18
0
50
1050.55
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
437.18
696.37
508.28
859.43
916.41
1178.53
1128.49
0
100
2: TOF MSMS 820.94ES+
9.21e3
226.12
100
750
200
300
400
500
600
700
800
m/z
900
1000
1100
1200
1300
1400
1500
MSMS ions search data
peak list (pkl)
Peptide mass : charge state : intensity
fragment mass : intensity
fragment mass : intensity
fragment mass : intensity
920.9598
241.0128
70.0629
72.0767
80.9474
110.0635
158.0875
173.1226
175.1129
185.0797
:
1769.7933
1771.8080
1839.8304
1841.9095
1843.8146
1845.8208
15.5793
22.1687
6.1025
8.3011
11.9145
71.9019
9.3308
7.5469
623.3281
243.3593
70.0612
71.0651
:
2
47.7946
43.3989
54.6593
44.5610
92.9938
58.4194
91.0550
3.5558
2
Mascot Search Overview
Mascot is a search engine which
uses mass spectrometry data to
identify proteins from primary
sequence databases
MASCOT provides 3 different search methods
• Peptide Mass Fingerprint
peptide mass values
• Sequence Query
peptide mass data plus amino acid sequence/composition
• MS/MS Ion Search
uninterpreted MS/MS data from one or more peptides
Cut-off score
for significance
is different for
every search
Protein name
Protein score
Number of matching peptides
Peptide score
Expect value
Different
species
Predicted
mass and
predicted pI
Sequence coverage
A match is made by correlating the
observed and predicted peptide masses
and their fragment ion masses –
the peptides themselves have not
actually been sequenced
All these proteins are hit #1
All have the same score and the same peptide masses match
The order of the list within each hit, is meaningless
i.e. cow is “top” here, but the sample is mouse
INSTRUCTIONS FOR THE PRACTICAL
COPY the 4 MSMS files onto the desktop
Click on the MS/MS Ions Search tool page
Standard search
Input your name & your e-mail
Use these standard defaults
swissprot
trypsin, 1 missed cleavage
variable on Carbamidomethyl C
variable on Oxidation M
peptide charge +2, +3, +4
Browse to add an MSMS file to the
search page
Micromass PKL
ESI-QUAD-TOF
Vary some parameters in
subsequent searches
try NCBInr and swissprot
databases for MSMS3
add in variable phosphorylations
for MSMS4
semi-trypsin for MSMS2
alter mass tolerances
compare results with standard
search
links
http://view6.workcast.net/?pak=3003276531895477&cpak=7053452047055213 MASCOT video tutorials
http://www.ionsource.com/ Mass Spectrometry and Biotechnology Resource –
lots of useful info – tutorials on de novo sequencing etc
http://proteome.nih.gov
proteomics special interest group at NIH,
includes archived videocasts of research seminars
http://ca.expasy.org/tools/ informatics tools e.g. peptidemass predicted digestion fragment tool
http://www.bspr.org/
British Society for Proteome Research
http://www.bmss.org.uk/
British Mass Spectrometry society
http://www.plasmaproteome.org/ The Plasma Proteome Institute in Washington D.C.
http://www.unimod.org/ Unimod : protein modifications for mass spectrometry
http://www.hupo.org/
http://www.spectroscopynow.com/coi/cda/home.cda?chId=0
http://www.abrf.org/index.cfm/group.show/Proteomics.34.htm
De novo sequencing
771+2
30SEPT05MS_ENOLASE1_771 MaxEnt 3 22 [Ev-150620,It50,En1] (0.050,2,Cmp)
R
M
E
L
1: TOF MSMS 771.36ES+
A
E
H
V
G
T
S
A
G
S
P
yMax
1541.76(M+H) +
100
%
885.46
y7
306.17
y2
242.12
b3
70.07
a1
P
175.13
y1
313.16
b4
619.35
y5
400.20 435.21
b5
y3
157.10
a2
748.39
y6
629.33
a8 679.32
548.30
522.24 y4
1041.54
y9
771.45
794.39
b9
886.53
984.51
y8
1142.60
y10
1042.62
1229.63
y11
1193.59
1300.65
y12
1357.70
y13
1444.71
y14
1426.68
1542.89
1523.76
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
Sequence reads in N to C direction - PSGASTGVHEAMR
1500
1543.59
M/z
1600
Example of good quality peptide match
Number of contiguous residues should be 5 or more
Have 8 for this peptide – good quality match
Example of poor quality peptide
Longest stretch of contiguous reside calls is 2 – insufficient for good ID
If this was the only peptide match it would be rejected by the user