Mascot Example Slides

1
Introduction to mass spectrometry-based protein
identification and quantification
Austin Yang, Ph.D.
Aebersold R, Mann M. Mass spectrometry-based
proteomics. Nature. 2003 Mar 13422(6928)198-207.
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.
2008 Jan7(1)51-61.
2
The typical proteomics experiment consists of
five stages
3
Mass spectrometers used in proteome research.
4
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
5
Electrospray Ionization (ESI)

• Multiple charging
• More charges for larger molecules
• MW range gt 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)
6
(No Transcript)
7
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
Relative Intensity
m/z
(464.29)
8
Peptide Sequencing by LC/MS/MS
9
Web addresses of some representative internet
resources for protein identification from mass
spectrometry data
10
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

11
SEQUEST Algorithm
Theoretical MS/MS spectra
Step 1. Determine Parent Ion molecular mass
Step 2.
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.
(Experimental MS/MS Spectrum)
Unified Scoring Function
12
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

Peptide 4
5 correct ()
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
13
Quantitative Mass Spec Analysis

• 1. Relative Quantitation
• a. ICAT Isotope-Coded Affinity Tags
• b. Digestion with Oxygen-18 Water
• c. Spectra Counting and Non-labeling
• Methodology
• 2. Absolute Quantitation

14
(No Transcript)
15
Alkylation of Cysteine Residue
Cysteine C3H5NOS 103.00918
Carboxymethyl Cys C5H7NO3S 161.01466

58.00548
16
Mascot Example Slides
ICAT
17
Trypsin Digestion with Oxygen18 and Oxygen16 Water
18
Absolute Quantification
Johri et al. Nature Reviews Microbiology 4, 932
942 (December 2006) doi10.1038/ nrmicro1552
19
Public Web Serverhttp//www.matrixscience.com/sea
rch_form_select.html Class Data
Downloadhttp//134.192.153.220/GPLS716Local
Web Serverhttp//134.192.153.220/mascotUsername
GPLS716Password GPLS716
20
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

21
Quantification Search Example

• 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
• Quantification Method 18O corrected multiplex

22
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_he
  lp.html

23
MS2 mixture example

• Data mixture10spectra.mgf
• Database yeast
• Fixed modifications Carbamidomethyl (C57.02)
• Variable modifications Oxidation (M)
• Peptide Mass Tolerance 0.1 Da
• Fragment Mass Tolerance 0.1 Da

24
Home Work

• 1. You will have to download your datasets from
  the following
• urlhttp//134.192.153.220/GPLS716
• a. Identification of phosphorylation site
  DataBIG3021307.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_1to5_071013.RAW.mgf.
• Submit your search results in pdf or html format
  to the following email address
  proteomicsumb_at_gmail.com 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
  ywan_at_som.umaryland.edu
• Phone number 8-2031