1. Mueller LN, Brusniak MY, Mani DR, Aebersold R
Introduction to mass spectrometry-based protein identification and quantification
Austin Yang, Ph.D.
Aebersold R, Mann M.
Mass spectrometry-based proteomics.
Nature Mar 13;422(6928): Review.
Mueller LN, Brusniak MY, Mani DR, Aebersold R
An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data.
J Proteome Res Jan;7(1):51-61.

2. The typical proteomics experiment consists of five stages

3. Mass spectrometers used in proteome research.

4. Monoistopic Mass =
Average Mass = (calculated)
As shown in Figure 1. the monoisotoptic mass of this compound is 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.

6. Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes%
A+1
A+2
A+3
A+4
12C 12
98.93(8)
13C
(10)
1.07(8)
14C
(4) - 1H
(4)
(70) 2H
(4)
0.0115(70) 3H
(11)
14N
(9)
99.632(7)
15N
(9)
0.368(7)
16O
(15)
99.757(16)
17O
(22)
0.038(1)
18O
(9)
0.205(14)
32S
(12)
94.93(31)
33S
(12)
0.76(2)
34S
(11)
4.29(28)
36S
(25)
0.02(1) .
19F
(7)
100
23Na
(23)
39K
(3)
(44)
40K
(29)
0.0117(1)
41K
(28)
6.7302(44)
31P
(20)
35Cl
(4)
75.781(4)
37Cl
(5)
24.22(4)
55Mn
(14)
54Fe
(14)
5.845(35)
56Fe
(15)
91.754(36)
57Fe
(15)
2.119(10)
58Fe
(15)
0.282(4)
63Cu
(15)
69.17(3)
65Cu
(19)
30.83(3)
79Br
(20)
50.69(7)
81Br
(3)
49.31(7)
127I
(4)

7. Peak Abundance, “Mass Crossover” and Calibration

8. The Nobel Prize in Chemistry 2002
"for the development of methods for identification and structure analyses of biological macromolecules"
"for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules"
John Fenn
Koichi Tanaka

9. Mass Spectrometry: A method to “weigh” molecules
Other information can be inferred from a weight measurement.
Post-translational modifications
Molecular interactions
Shape
Sequence
Physical dimensions
etc...
A simple measurement of mass is used to confirm the identity of a molecule, but it can be used for much more……

10. Matrix-assisted Laser Desorption/Ionization (MALDI)
Time-of-Flight (TOF) Analyzer
detector
high voltage v1 m1 v2 m2 v3 m3
MALDI
sample
laser
drift region
m1 m2 m3

11. Electrospray: Generation of aerosols and droplets
“Wings to Molecular Elephants”

12. Electrospray Ionization (ESI)
Multiple charging
More charges for larger molecules
MW range > 150 kDa
Liquid introduction of analyte
Interface with liquid separation methods, e.g. liquid chromatography
Tandem mass spectrometry (MS/MS) for protein sequencing
ESI MS
high voltage
highly charge droplets
20+
19+
18+
21+
17+
16+
22+
15+
14+
500
700
900
1100
mass/charge (m/z)

13. Origin of the ES Spectra of Peptides4+ 3+ 2+ 1+
m/z = (Mr+4H)/4
m/z = (Mr+3H)/3
m/z = (Mr+2H)/2
m/z = (Mr+H)
Rel. Inten.
m/z
ES-MS

14. Theoretical CID of a Tryptic Peptide+ + + + F L G K + + F L G K b3 y1 F L G K + +
Parent
ions + + F L G K F L G K
CID b2 y2 + F L G K + + + + F L G K F L G K b1 y3
Non-dissociated
Parent ions
Daughter ions y1 y2 y3 b1 b2 b3 K G L F
MS/MS
Spectrum
Relative Intensity L F G K
m/z
(464.29)

15. Peptide Sequencing by LC/MS/MS

16. Web addresses of some representative internet resources for protein identification from mass spectrometry data

17. Data Mining through SEQUEST and PAULA
Database Search Time
Yeast ORFs (6,351 entries) sec: sec/s
Non-redundant protein (100k entries) min:
EST (100K entries, 3-frames) ,000 min:

18. Determine Parent Ion molecular mass
SEQUEST Algorithm
Theoretical MS/MS
spectra
Step 1.
Determine Parent Ion molecular mass
Step 2.
STEP 1.
SEQ 1
SEQ 2
SEQ 3
SEQ 4
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)
ZSA-charge assignment
Step 4.
Scores are ranked and
Protein Identifications are made based on these cross correlation scores.
Step 3.
Experimental Spectrum is compared with each theoretical spectra and correlation scores are assigned.
STEP 3.
Unified Scoring Function
(Experimental MS/MS Spectrum)

19. Amplification of False Positive Error Rate from Peptide to Protein Level+
Prot A
Peptide 1
in the sample
(enriched for ‘multi-hit’ proteins)
Peptide 2
Prot B +
Peptide 3 + 5
correct
(+)
Peptide 4
Peptide 5
Prot
Peptide 6
not in the
sample
(enriched for ‘single hits’) +
Peptide 7
Prot
Prot
Peptide 8
Prot
Peptide 9
Prot +
Peptide10
Peptide Level: 50% False Positives
Protein Level: 71% False Positives

20. Quantitative Mass Spec Analysis
1. Relative Quantitation
a. SILAC and iTRAQ
b. Digestion with Oxygen-18 Water
c. Spectra Counting and Non-labeling
Methodology
2. Absolute Quantitation

22. Trypsin Digestion with Oxygen18 and Oxygen16 Water

24. Limitation of SILAC

25. Multiplexed Isobaric Tagging Technology (iTRAQ)
Philip L. Ross, et al. Molecular & Cellular Proteomics 3:1154–1169, 2004.

26. Release of 114 and 117 Reporter Ions
Parent Ion
Regular CID to obtain sequence
Low mass cut-off and no reporter ion
High Energy Collision Cell
to quantify and sequence

27. Protein name 117/114 ratio Num of pep PSD93 2.829 5 PSD95 2.021 21
Loading 10ug
9 salt cuts online 2D_LC_MS/MS
962 proteins are quantified
Protein name
117/114 ratio
Num of pep
PSD93
2.829 5
PSD95
2.021 21
PSD95-AP1
1.764 2
GABA alpha
1.365
GABA beta
2.087 3
NR2B
1.813 4
AMPA1
2.092 7
AMPA2
1.921 11
AMPA4
1.902
NR1
1.658 6
Expected ratio

28. Absolute Quantification
Johri et al. Nature Reviews Microbiology 4, 932 – 942 (December 2006) | doi: / nrmicro1552

30. Public Web Server http://www. matrixscience. com/search_form_select
Public Web Server Class Data Download: Local Web Server Username: GPILS
Password: GPILS

31. 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

32. Quantification Search Example
Note: Save link as; Save this file to the desktop)
Data: 18O_BSA_100fmol_1to5_01_ 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 32

33. MS/MS Database Search Example
Data: BSA onespectra.mgf (one spectra)
Database: bovine
Fixed modifications: Carboxymethyl(C )
Varied modifications: Oxidatation(M)
Peptide Mass Tolerance : 0.1 Da
Fragment Mass Tolerance: 0.1 Da

34. Alkylation of Cysteine Residue
Cysteine C3H5NOS
Carboxymethyl Cys C5H7NO3S

35. 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

36. Home Work
1. You will have to download your datasets from the following
url:
a. Identification of phosphorylation site : Data:BIG 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_ RAW.mgf.
Submit your search results in pdf or html format to the following address: Please include the following information when you submit your homework
1. Your name and ID in the subject of your
2. Search parameters
3. A short summary of your search results.
Questions: Contact Yunhu Wan,
Phone number: