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