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My contact details and information about submitting samples for MS
http://www.nottingham.ac.uk/Biosciences/People/susan.liddell
We use 2 types of MS for PROTEIN IDENTIFICATION
• PEPTIDE MASS FINGERPRINTING (PMF)
– MALDI-ToF MS
• TANDEM MS (aka MSMS)– ELECTROSPRAY Q-ToF2
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
Protein digestion
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)
This is unique to the protein and can be used to identify it 95.4
89.3112.1
105.3 = 402.2
95.4
89.3
112.1
105.3
97.1101.8
= 601
89.3 = 89.3
Laser
energy Peptide ions enter the
Time of Flight tube
separated on basis of mass
Peptides co-crystallised with matrixIonise peptides
Detector
mass/charge of every peptide
peptide mass fingerprint
MALDI-TOF-MS (Matrix Assisted Laser Desorption and Ionisation)
peptide mass spectrum
a trypsin digest of a single protein every peak corresponds to the mass (m/z) of a peptide ion
m / z = mass / charge
relativeintensity
Peptide mass spectrum
Data converted to text
List of peptide masses
. . . .
1051.541094.561244.641476.671542.841613.881664.971763.791952.892264.892238.23 . . . .
a peak list (pkl)
=fingerprintfingerprint
run digested protein on MS
Database searches with PeptideMassFingerprint data
sequence databases
theoretical trypsin digest of every
predicted protein
list of calculated peptide masses
compare
identification made if match
is found
list of measured peptide masses
“fingerprint”
. . .1051.541094.561244.641476.671542.841613.881664.971763.791952.892264.892238.23 . . .
peptide mass fingerprinting• rapid
• high throughput
• large scale identification of proteins from organisms whose
genomes are completely sequenced
• good tool for a first look at a sampleBUT…….
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 - tandem MS
Samples in solution
Compatible with HPLC
Complex protein mixtures
Determine peptide masses
Peptide fragmentation
Peptide sequence
ElectroSpray Ionisation (ESI) Mass Spectrometry on the Q-ToF2
MS2MS1
MS on the Q-ToF2
Tandem MS - peptide fragmentation
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
low energy collision fragments the peptide
cleavage usually occurs at the amide bond i.e. between residues
peptide fragmentation
the series of fragments corresponds to the sequence of the peptide
M/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
100
strong, approx. 80 kDa 1D gel bandGUY_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+
1028.65y10
870.58y8313.20
b3242.16b2
214.16a2
147.12y186.10
L
757.50y7
642.47y6
426.28b4409.25 537.32
554.34b5
677.37
782.44
791.43
941.63y9
906.451010.64
1563.98
1156.73y11
1139.69
1138.741029.75
1340.85y13
1269.81y12
1191.67 1546.931341.74
De novo sequencing
M/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600
%
0
100
771+230SEPT05MS_ENOLASE1_771 MaxEnt 3 22 [Ev-150620,It50,En1] (0.050,2,Cmp) 1: TOF MSMS 771.36ES+
R M E L A E H V G T S A G S P yMax1541.76(M+H) +
885.46y7
306.17y2
242.12b3
175.13y1
70.07a1P
157.10a2
619.35y5
313.16b4
435.21y3
400.20b5 548.30
y4522.24
771.45748.39y6
629.33a8 679.32
794.39b9
1041.54y9
984.51y8886.53
1229.63y11
1142.60y10
1042.62 1193.59
1444.71y141357.70
y131300.65y12 1426.68
1523.76
1542.89
1543.59
Sequence reads in N to C direction - PSGASTGVHEAMR
m/z400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550
%
0
100 582.28
476.25
951.45
708.40837.45
810.74738.85
1163.59
1050.55
Survey Mass Spectrum (MS) - intact peptides detected in a 1 second survey scan
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
100
1F_MP0709_05MAY09 169 (37.007) Cm (167:196) 4: TOF MSMS 777.82ES+ 1.28e3662.81
235.12
147.12
207.13
409.22
262.14
306.16
614.27
465.17
437.18
536.22
508.28
1090.52
1019.48
778.33
696.37
859.43916.41 1178.53
1128.49
MSMS
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
1F_MP0709_05MAY09 194 (37.239) Cm (191:217) 2: TOF MSMS 820.94ES+ 9.21e3226.12
129.11
900.51
325.19
262.16
706.89
361.22490.26
589.34820.97
1088.59
1001.57
1187.67
1412.79
1315.76
MSMS
On-line LC-MSMS on the Q-ToF2 : peptides from a single protein
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
1F_MP0709_05MAY09 190 (39.732) Cm (190:216) 3: TOF MSMS 788.86ES+ 1.58e3129.10
1123.53
242.19
668.31562.26
294.18
454.27
789.40
732.35913.42
1026.48
1335.59
1238.56
Fragment Mass spectrum (MSMS)fragments from one peptide
MSMS
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200
%
0
100
1F_MP0709_05MAY09 272 (45.212) Cm (271:290) 2: TOF MSMS 582.28ES+ 3.92e3185.17
147.12
951.48
595.31365.22213.16
326.18
494.27
440.23 569.27
708.41
837.46
1050.55
MSMS
Many peptides are fragmented during a 60 minute runLC-MSMS generates much more data than fingerprinting
mass of intact peptides & the fragment massesSearch databases with much more data per protein
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 270.0629 15.579372.0767 22.168780.9474 6.1025110.0635 8.3011158.0875 11.9145173.1226 71.9019175.1129 9.3308185.0797 7.5469:1769.7933 47.79461771.8080 43.39891839.8304 54.65931841.9095 44.56101843.8146 92.99381845.8208 58.4194
623.3281 243.3593 270.0612 91.055071.0651 3.5558:
Examples of open access search tools
MASCOT3 types of searcheshttp://www.matrixscience.com/search_form_select.html
AldentePMF search tool
http://ca.expasy.org/tools/aldente/
Phenyx an MS data analysis platform
identification and characterization of proteins & peptides from mass spectrometry data
http://ca.expasy.org/tools/aldente/
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
Decoy database search
Peptide score
Expect value
Number of matching peptidesProtein scoreProtein name
Different species
Only the peptide masses and their fragment ion masses are matched – the peptides themselves have not
actually been sequenced
Predicted mass and predicted pI
Sequence coverage
All these proteins are hit #1All have the same score and the same peptide masses matchThe order of the list within each hit, is meaningless i.e. cow is “top” here, but the sample is mouse
Download the MSMS files 1 to 4 onto the desktop
Click on the MS/MS Ions Search tool page
Standard search
input your name & your e-mail
use standard defaults swissprot trypsin, 1 missed cleavage variable on Carbamidomethyl C variable on Oxidation M peptide charge +2, +3, +4
Copy MSMS files to desktopBrowse to add file to 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
alter mass tolerances
compare results with standard search
Selected references and reviews
Gorg A, Weiss W, Dunn MJ.Current two-dimensional electrophoresis technology for proteomics.Proteomics. 2004 Dec;4(12):3665-85.
Two-dimensional gel electrophoresis: an overview, Pages 263-272 David E. GarfinTrends in Analytical Chemistry Volume 22, Issue 5, Pages 263-334 (May 2003)
The current state of two-dimensional electrophoresis with immobilized pH gradients.Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. Electrophoresis. 2000 Apr;21(6):1037-53.
ANALYSIS OF PROTEINS AND PROTEOMES BY MASS SPECTROMETRYMatthias Mann, Ronald C. Hendrickson, and Akhilesh Pandey Annual Review of Biochemistry July 2001, Vol. 70, pp. 437-473
Challenges in mass spectrometry-based proteomics. Reinders J, Lewandrowski U, Moebius J, Wagner Y, Sickmann A. Proteomics. 2004 Dec;4(12):3686-703.
Plant proteome analysis.Canovas FM, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock HP, Rossignol M. Proteomics. 2004 Feb;4(2):285-98.
Subcellular proteomics.Dreger M.Mass Spectrom Rev. 2003 Jan-Feb;22(1):27-56.
Functional organization of the yeast proteome by systematic analysis of protein complexes.Gavin AC, Bosche M, et al Nature. 2002 Jan 10;415(6868):141-7.
Development. 2004 Feb;131(3):643-656. Drosophila ventral furrow morphogenesis: a proteomic analysis.Lei Gong, Mamta Puri, Mustafa Ünlü, Margaret Young, Katherine Robertson, Surya Viswanathan, Arun Krishnaswamy, Susan R. Dowd and Jonathan S. Minden
State-of-the-art in phosphoproteomicsProteomics 2005 Early View i.e. find it on the journals early view section of the web site Joerg Reinders, Albert Sickmann
Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF.Proteomics. 2003 Jul;3(7):1128-44.
Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing.Nat Rev Mol Cell Biol. 2004 Sep;5(9):699-711. Review.
linkshttp://www.swissproteomicsociety.org/digest Swiss Proteomics Society. The “digest” provides a consolidated selection of articles published in all scientific
publications that are pertinent to proteomics – finds all the interesting and relevant papers for you!
http://proteome.nih.gov proteomics special interest group at NIH, includes archived videocasts of research seminars
http://ca.expasy.org/tools/ proteome 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.ionsource.com/ Mass Spectrometry and Biotechnology Resource – lots of useful info – tutorials on de novo sequencing etc
http://www.abrf.org/index.cfm/group.show/Proteomics.34.htm
Example of good quality peptide match
Number of contiguous residues should be 5 or moreHave 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