Mascot Example Slides

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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 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.
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The typical proteomics experiment consists of
five stages
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Mass spectrometers used in proteome research.
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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
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Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes
Iso A Iso A1 Iso A2 Iso A3 Iso A4
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) - - - 36S 35.96708088(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) 58Fe 57.9332805(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 - - - - - - - - - - - -
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Peak Abundance, Mass Crossover and Calibration
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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"
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Mass SpectrometryA method to weigh molecules
A simple measurement of mass is used to confirm
the identity of a molecule, but it can be used
for much more
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Matrix-assisted Laser Desorption/Ionization
(MALDI)
Time-of-Flight (TOF) Analyzer
detector
high voltage
MALDI
sample
laser
drift region
m1 m2 m3
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Electrospray Generation of aerosols and droplets
Wings to Molecular Elephants
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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
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18
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16
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500
700
900
1100
mass/charge (m/z)
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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)
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Peptide Sequencing by LC/MS/MS
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Web addresses of some representative internet
resources for protein identification from mass
spectrometry data
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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

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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
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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
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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

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Trypsin Digestion with Oxygen18 and Oxygen16 Water
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Limitation of SILAC
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Multiplexed Isobaric Tagging Technology (iTRAQ)
Isobaric Tag 145
Philip L. Ross, et al. Molecular Cellular
Proteomics 311541169, 2004.
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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
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PSD_117 PSD_11421 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 2
GABA beta 2.087 3
NR2B 1.813 4
AMPA1 2.092 7
AMPA2 1.921 11
AMPA4 1.902 4
NR1 1.658 6
Expected ratio
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Absolute Quantification
Johri et al. Nature Reviews Microbiology 4, 932
942 (December 2006) doi10.1038/ nrmicro1552
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Public Web Serverhttp//www.matrixscience.com/sea
rch_form_select.html Class Data
Downloadhttp//10.90.157.112/GPLS716Local Web
Serverhttp//10.90.157.112/mascotUsername
GPILS Password GPILS
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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

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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

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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

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Alkylation of Cysteine Residue
Cysteine C3H5NOS 103.00918
Carboxymethyl Cys C5H7NO3S 161.01466

58.00548
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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

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Home Work

• 1. You will have to download your datasets from
  the following
• urlhttp//10.90.157.112/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_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