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Proteomics and mass spectrometry

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M = mass of the peak of interest. ? m = width in daltons of the peak ... on the size distribution of peptide masses as a function of protein mass. ... – PowerPoint PPT presentation

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Title: Proteomics and mass spectrometry


1
Proteomics and mass spectrometry
  • Manimalha Balasubramani

2
Outline
  • Mass spectrometers
  • Protein identification
  • Quantitative proteomics
  • Protein-protein interactions

3
A mass spectrum
4
Basically measures mass
Adapted from google
5
Components
Adapted from an Analytical chemistry textbook
6
Ionization process
Matrix Assisted Laser Desorption Ionization
MALDI
ElectroSpray Ionization
ESI
Nobel prize in Chemistry, 2002
7
MALDI Matrix Assisted Laser Desorption
Ionization
8
ESI Electro Spray Ionization
9
Mass analyzers several designs
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
10
GPCL inventory
  • ABI Voyager DE PRO, walk-up use
  • ABI 4700 Proteomics Analyzer
  • Thermoelectron LCQ Deca with Surveyor HPLC
  • ABI Qstar Elite with Ultimate 3000 HPLC
  • Bruker micrOTOF with Ultimate 3000 HPLC
  • Bruker 12 Tesla FTMS with Ultimate 3000 HPLC

11
Time-of-flight (TOF) analyzers
MALDI TOF Voyager DE PRO
ESI TOF Ultimate 3000 with micrOTOF
12
MALDI TOF - principle
KE zeV 1mv2 2
13
MS of serum albumin
ESI TOF
MALDI TOF
14
Tandem mass spectrometer
MALDI TOF/TOF
MS and MS/MS
15
Ion Trap
MS, MS2, MS3, .MSn
16
Quadrupole-q-TOF
ESI QqTOF
17
installation phase.
FT MS
18
bottom line
..Resolution and mass accuracy
19
FWHM
  • Full width at half maxima of a peak

20
Resolution and mass accuracy
?m measured at 50 peak height is the Full Width
at Half Maxima (FWHM)
R M ?m R resolution M
mass of the peak of interest ? m width
in daltons of the peak
21
Mass accuracy is measured as parts per million
value
ppm 106?m 106 M
R
22
outline
  • Mass spectrometers
  • Protein identification
  • Quantitative proteomics
  • Protein-protein interactions

23
Peptide Mass Fingerprinting - PMF
Database entry NCBI
From http//gobi.ym.edu.tw/course/mass/2004-0325.
pdf
24
Informatics
  • Search engines
  • Mascot, Matrix Science
  • Sequest, Thermoelectron
  • Free-ware
  • Protein prospector (http//prospector.ucsf.edu/)
  • TPP tools (http//tools.proteomecenter.org/TPP.php
    )

25
Database searching using MASCOT Overview of the
experiment Submission of data to MASCOT
webserver
26
1D SDS PAGE of proteins
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
27
Mass spectrum
Intensity
Mass to charge ratio (m/z)
28
Peak list
  • Compiled from the mass spectra
  • Mass list
  • Mass list and intensity
  • Submitted to the search engine

29
http//www.matrixscience.com/
30
Mascot scoring
31
List of common contaminants
  • Trypsin autolysis peptides
  • Matrix peaks
  • Keratin from skin, hair
  • Other contaminants

32
Protein Identification
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
33
Tandem mass spectrum
http//qbab.aber.ac.uk
34
Tandem mass spectrum
35
Tandem mass spectra (MS/MS) can be used for
peptide sequencing
  • Database Searching
  • Peptide Mass Fingerprinting
  • Sequence tag approach
  • De novo sequencing
  • inspect raw data

http//qbab.aber.ac.uk
36
(No Transcript)
37
Top hits from Mascot Search there are multiple
accession numbers for the same protein
38
Search returns a cluster of proteins with the
same matching peptides
39
Creatine kinase B is the highest scoring protein
Match to gi21536286 Score 681
Creatine kinase - B Homo sapiens
Nominal mass (Mr) 42591 Calculated pI value
5.34 Observed Mass pI 43kd, 6.2-6.27
Sequence Coverage 46
1 MPFSNSHNAL KLRFPAEDEF PDLSAHNNHM AKVLTPELYA ELRAKSTPSG
51 FTLDDVIQTG VDNPGHPYIM TVGCVAGDEE SYEVFKDLFD PIIEDRHGGY
101 KPSDEHKTDL NPDNLQGGDD LDPNYVLSSR VRTGRSIRGF CLPPHCSRGE
151 RRAIEKLAVE ALSSLDGDLA GRYYALKSMT EAEQQQLIDD HFLFDKPVSP
201 LLSASGMARD WPDARGIWHN DNKTFLVWVN EEDHLRVISM QKGGNMKEVF
251 TRFCTGLTQI ETLFKSKDYE FMWNPHLGYI LTCPSNLGTG LRAGVHIKLP
301 NLGKHEKFSE VLKRLRLQKR GTGGVDTAAV GGVFDVSNAD RLGFSEVELV
351 QMVVDGVKLL IEMEQRLEQG QAIDDLMPAQ K
40
outline
  • Mass spectrometers
  • Protein identification
  • Quantitative proteomics
  • Protein-protein interactions

41
Quantitative Proteomics
Sample preparation
42
From 2D gels .to MALDI or ESI MS
Control Test
Pool
Cy3 Cy5
Image analysis with Delta2D, Decodon Quantitate
Export spot list to robotic picker
43
..its high-throughput
1st Dimension - Isoelectric focussing
2nd Dimension SDS PAGE
Spot picking
Trypsin gel digest
44
Colorectal cancer markers
Balasubramani et al., Cancer Res., 2006
45
Shotgun proteomics
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
46
typical workflow to identify biomarkers that
distinguish indolent versus aggressive forms of
cancer..
47
Sample handling
In-solution Isoelectric focussing
1D or 2D LC MALDI
HPLC
48
Protein-protein interaction studies
  • Immunoaffinity pull-downs
  • Tandem affinity purification

49
GPCL
  • Billy W Day
  • Paul Wood
  • Mirunalni Thangavelu
  • Tamanna Sultana
  • Emanuel M Schreiber
  • Chris Bolcato
  • Chris Myers
  • Patrick Miller
  • Robert Wolfe

50
definitions
  • The amu is defined as 1/12th the mass of one
    neutral 6C12 atom
  • Amu is also called the dalton
  • 1 amu 1/12 ( 12g 12C/mol 12C
  • 6.0221 x 1023 atoms 12C/mol 12C
  • 1.6605 x 10-24 g/atom 12C

51
Isotopic species of M
  • (M H) (M 1H)/1H
  • (M 2H)2 (M 2H)/2H
  • (M 3H)3 (M 3H)/3H
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