introduction to mass spectrometry- based protein identification and quantification austin yang,...
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Introduction to mass spectrometry-based protein identification and quantification
Austin Yang, Ph.D.
Aebersold R, Mann M.Mass spectrometry-based proteomics.Nature. 2003 Mar 13;422(6928):198-207. Review.
Mueller LN, Brusniak MY, Mani DR, Aebersold RAn assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data.J Proteome Res. 2008 Jan;7(1):51-61.
The typical proteomics experiment consists of five stages
Mass spectrometers used in proteome research.
Monoistopic Mass = 1155.6
Average Mass = 1156.3 (calculated)
As shown in Figure 1. the monoisotoptic mass of this compound is 1155.6. For a given compound the monoisotopic mass is the mass of the isotopic peak whose elemental composition is composed of the most abundant isotopes of those elements. The monoisotopic mass can be calculated using the atomic masses of the isotopes.
The average mass is the weighted average of the isotopic masses weighted by the isotopic abundances. The average mass can be calculated using the atomic weights of the elements.
www.ionsource.com
Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes
Iso
A %Iso
A+1 %Iso
A+2 %Iso
A+3 %Iso
A+4 %
12C
12 98.93(8)13C
13.0033548378(10) 1.07(8)14C
14.003241988(4) - - - - - - -
1H 1.0078250321(4) 99.9885(70)2H
2.0141017780(4)0.0115(
70)3H 3.0160492675(11) - - - - - - -
14N
14.0030740052(9) 99.632(7)15N
15.0001088984(9) 0.368(7) - - - - - - - - -
16O
15.9949146221(15) 99.757(16)17O
16.99913150(22) 0.038(1)18O
17.9991604(9) 0.205(14) - - - - - -
32S 31.97207069(12) 94.93(31)33S
32.97145850(12) 0.76(2)34S
33.96786683(11) 4.29(28) - - -36
S35.96708088(25) 0.02(1)
. . . . . . . . . . . . . . .
19F 18.99840320(7) 100 - - - - - - - - - - - -
23Na
22.98976967(23) 100 - - - - - - - - - - - -
39K
38.9637069(3) 93.2581(44)40K
39.96399867(29)0.0117(
1)41K
40.96182597(28)6.7302(44
)- - - - - -
31P 30.97376151(20) 100 - - - - - - - - - - - -
35Cl
34.96885271(4) 75.781(4) - - -37Cl
36.96590260(5) 24.22(4) - - - - - -
55Mn
54.9380496(14) 100 - - - - - - - - - - - -
54Fe
53.9396148(14) 5.845(35) - - -56Fe
55.9349421(15)91.754(36
)
57Fe
56.9353987(15)2.119(10)
58
Fe57.9332805(15)
0.282(4)
63Cu
62.9296011(15) 69.17(3) - - -65Cu
64.9277937(19) 30.83(3) - - - - - -
79Br
78.9183376(20) 50.69(7) - - -81Br
80.916291(3) 49.31(7) - - - - - -
127I
126.904468(4) 100 - - - - - - - - - - - -
Peak Abundance, “Mass Crossover” and Calibration
The Nobel Prize in Chemistry 2002The Nobel Prize in Chemistry 2002
"for the development of methods for identification and "for the development of methods for identification and structure analyses of biological macromolecules" structure analyses of biological macromolecules"
"for their development of soft desorption ionisation "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological methods for mass spectrometric analyses of biological macromolecules"macromolecules"
John FennJohn Fenn Koichi TanakaKoichi Tanaka
Mass Spectrometry:Mass Spectrometry:A method to “weigh” moleculesA method to “weigh” molecules
A simple measurement of A simple measurement of mass is used to confirm the mass is used to confirm the identity of a molecule, but it identity of a molecule, but it can be used for much can be used for much more……more……
Other information can be inferred Other information can be inferred from a weight measurement.from a weight measurement.
• Post-translational modificationsPost-translational modifications• Molecular interactionsMolecular interactions• ShapeShape• SequenceSequence• Physical dimensionsPhysical dimensions• etc...etc...
high voltagehigh voltage
samplesample
vv11
m1
vv22
m2
vv33
m3
m1 m2 m3
detectordetector
drift regionlaserlaser
Matrix-assisted Laser Matrix-assisted Laser Desorption/Ionization (MALDI)Desorption/Ionization (MALDI)
Time-of-Flight (TOF) AnalyzerTime-of-Flight (TOF) Analyzer
MALDIMALDI
Electrospray: Generation of aerosols and droplets
“Wings to Molecular Elephants”
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
• Multiple chargingMultiple charging– More charges for larger moleculesMore charges for larger molecules
• MW range > 150 kDaMW range > 150 kDa• Liquid introduction of analyteLiquid introduction of analyte
– Interface with liquid separation Interface with liquid separation methods, e.g. liquid methods, e.g. liquid chromatographychromatography
– Tandem mass spectrometry Tandem mass spectrometry (MS/MS) for protein sequencing(MS/MS) for protein sequencing
500500 700700 900900 11001100
mass/charge (mass/charge (m/zm/z))
20+20+19+19+
18+18+
17+17+
16+16+
15+15+14+14+
21+21+
22+22+
highly charge highly charge dropletsdroplets
MSMS
ESIESI
Origin of the ES Spectra of PeptidesOrigin of the ES Spectra of Peptides
H
H
HH
4+
HH
H
3+
H
2+
H
1+
H
m/z = (Mr+4H)/4
m/z = (Mr+3H)/3
m/z = (Mr+2H)/2
m/z = (Mr+H)
1+
2+
3+
4+
Rel. Inten.
m/z
ES-MS
b1
b2
b3
y1
y2
y3
LF G K
Rela
t ive I
nte
nsit
y
m/z
F L G K
++
F L G K
++
F L G K
++
CID
F L G K++
F L G K
++
F L G K
++
b1
b2
b3
y3
y2
y1 F L G K
++
F L G K
+
Theoretical CID of a Tryptic Peptide
K G L F
MS/MSSpectrum
Parentions
(464.29)
Daughter ionsNon-dissociatedParent ions
Peptide Sequencing by LC/MS/MS
Web addresses of some representative internet resources for protein identification from mass
spectrometry data
Program Web Address
BLAST http:/ / www.ebi.ac.uk/ blastall/
Mascot http:/ / www.matrixscience.com/ cgi/ index.pl?page=/ home.html
MassSearch http:/ / cbrg.inf .ethz.ch/ Server/ ServerBooklet/ MassSearchEx.html
MOWSE http:/ / srs.hgmp.mrc.ac.uk/ cgi-bin/ mowse
PeptideSearch http:/ / www.narrador.embl-
heidelberg.de/ GroupPages/ PageLink/ peptidesearchpage.html
Protein Prospector http:/ / prospector.ucsf .edu/
Prowl http:/ / prowl.rockefeller.edu/
SEQUEST http:/ / fields.scripps.edu/ sequest/
Data Mining through SEQUEST and PAULA
Database Search Time•Yeast ORFs (6,351 entries) 52 sec: 0.104 sec/s•Non-redundant protein (100k entries) 3500 min: •EST (100K entries, 3-frames) 5-10,000 min:
SEQ 1
SEQ 2
SEQ 3
SEQ 4
STEP 1.
STEP 3.
SEQUEST Algorithm
(Experimental MS/MS Spectrum)
500 peptides with masses closest to that of the parent ion are retrieved from a protein database. Computer generates a theoretical MS/MS Spectrum for each peptide sequence (SEQ1, 2, 3, 4, …)
(Experimental MS/MS Spectrum)
Theoretical MS/MSspectra
Step 1.Determine Parent
Ion molecular mass
Step 2.
Step 3.Experimental Spectrum is compared with each theoretical spectra and correlation scores are assigned.
Step 4.Scores are ranked andProtein Identifications are made based on these cross correlation scores.
ZSA-charge assignment
Unified Scoring Function
Prot APeptide 1
Peptide 2
Prot BPeptide 3
Peptide 4
Peptide 5
Prot
Prot
Prot
Prot
in the sample(enriched for ‘multi-hit’ proteins)
not in the sample(enriched for ‘single hits’)Prot
Peptide 6
Peptide 7
Peptide 8
Peptide 9
Peptide10
+
++
+
+
5correct (+)
Amplification of False Positive Error Rate from Peptide to Protein Level
Peptide Level: 50% False Positives
Protein Level: 71% False Positives
Quantitative Mass Spec Analysis
1. Relative Quantitationa. SILAC and iTRAQb. Digestion with Oxygen-18 Waterc. Spectra Counting and Non-labeling Methodology 2. Absolute Quantitation
Trypsin Digestion with Oxygen18 and Oxygen16 Water
Limitation of SILAC
Philip L. Ross, et al. Molecular & Cellular Proteomics 3:1154–1169, 2004.
Multiplexed Isobaric Tagging Technology (iTRAQ)
Isobaric Tag = 145
BSA114_115_21 #6272 RT: 33.88 AV: 1 NL: 1.99E6T: ITMS + c ESI d Full ms2 [email protected] [50.00-1965.00]
200 400 600 800 1000 1200 1400 1600 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative A
bundance
650.29
649.67
928.15371.76 653.27619.71 1437.901054.49 1154.70 1560.73 1647.55133.01
BSA114_117_31 #6446 RT: 34.78 AV: 1 NL: 1.58E5T: ITMS + c ESI d Full ms2 [email protected] [50.00-1965.00]
200 400 600 800 1000 1200 1400 1600 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tiv
e A
bundance
371.56
372.42
927.89
929.26
420.28 742.55578.07822.79291.36 635.59
145.131010.58 1435.841154.58 1251.79 1536.66 1711.79
BSA114_117_35 #6127 RT: 32.86 AV: 1 NL: 1.34E4T: ITMS + c ESI d Full ms2 [email protected] [50.00-1965.00]
200 400 600 800 1000 1200 1400 1600 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tiv
e A
bundance
928.57
420.15
742.65
291.14402.30
606.37114.05 735.56
878.72578.14
772.20145.06
228.20 1010.13 1430.54
1501.851034.501167.64
1566.86
Release of 114 and 117 Reporter Ions
Parent Ion
Regular CID to obtain sequenceLow mass cut-off and no reporter ion
High Energy Collision Cellto quantify and sequence
PSD_117: PSD_114=2:1Loading 10ug9 salt cuts online 2D_LC_MS/MS962 proteins are quantified
Expected ratio
Protein name117/114
ratioNum of
pep PSD93 2.829 5PSD95 2.021 21PSD95-AP1 1.764 2GABA alpha 1.365 2GABA beta 2.087 3NR2B 1.813 4AMPA1 2.092 7AMPA2 1.921 11AMPA4 1.902 4NR1 1.658 6
Johri et al. Nature Reviews Microbiology 4, 932 – 942 (December 2006) | doi:10.1038/ nrmicro1552
Absolute Quantification
Public Web Serverhttp://www.matrixscience.com/search_form_select.html
Class Data Download:http://10.90.157.112/GPLS716
Local Web Serverhttp://10.90.157.112/mascotUsername: GPILSPassword: GPILS
MS1 PMF(peptide mass fingerprinting) Search Example
• Data: testms1.txt, 210 MS1 peaks• Database: bovine• Fixed modifications : Carboxymethyl (C)
Variable modifications : Oxidation (M)• Peptide Tolerance: 0.1 Da• Monoisotopic mass• Mass Value: Mr
Quantification Search Example• Note: Save link as; Save this file to the desktop)• Data:
18O_BSA_100fmol_1to5_01_071018.RAW.mgf• Database: bovine• Fixed modifications : Carbamidomethyl (C)• Peptide Tolerance: 8 Da (required for O18 labeling)• Fragment Tolerance: 0.2 Da• Peptide Charge: Mr• Quantification Method: 18O corrected multiplex
MS/MS Database Search Example
• Data: BSA onespectra.mgf (one spectra)• Database: bovine• Fixed modifications: Carboxymethyl(C + 58.01)• Varied modifications: Oxidatation(M)• Peptide Mass Tolerance : 0.1 Da • Fragment Mass Tolerance: 0.1 Da • http://www.matrixscience.com/help/
fragmentation_help.html
Cysteine C3H5NOS 103.00918
Carboxymethyl Cys C5H7NO3S 161.01466 58.00548
Alkylation of Cysteine Residue
MS2 mixture example
• Data: mixture10spectra.mgf• Database: yeast• Fixed modifications : Carbamidomethyl (C+57.02) • Variable modifications : Oxidation (M)• Peptide Mass Tolerance : 0.1 Da • Fragment Mass Tolerance: 0.1 Da
Home Work1. You will have to download your datasets from the following url:http://10.90.157.112/GPLS716 a. Identification of phosphorylation site : Data:BIG3021307.RAW.mgf Recommend parameters: Database: human. Variable Modification: Phospho(ST) Fixed modification: Carboamidomethyl(C).
b. Quantificaiton of oxygen-18/oxygen-16 digested BSA
Data: 18O_BSA_500fmol_071013.RAW.mgf.
Submit your search results in pdf or html format to the following email address: [email protected]; Please include the following information when you submit your homework
1. Your name and ID in the subject of your email 2. Search parameters
3. A short summary of your search results.
Questions: Contact Yunhu Wan, email: [email protected] Phone number: 8-2031