Proteomics and mass spectrometry

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Proteomics and mass spectrometry

• Manimalha Balasubramani

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Outline

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

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A mass spectrum
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Basically measures mass
Adapted from google
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Components
Adapted from an Analytical chemistry textbook
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Ionization process
Matrix Assisted Laser Desorption Ionization
MALDI
ElectroSpray Ionization
ESI
Nobel prize in Chemistry, 2002
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MALDI Matrix Assisted Laser Desorption
Ionization
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ESI Electro Spray Ionization
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Mass analyzers several designs
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
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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

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Time-of-flight (TOF) analyzers
MALDI TOF Voyager DE PRO
ESI TOF Ultimate 3000 with micrOTOF
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MALDI TOF - principle
KE zeV 1mv2 2
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MS of serum albumin
ESI TOF
MALDI TOF
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Tandem mass spectrometer
MALDI TOF/TOF
MS and MS/MS
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Ion Trap
MS, MS2, MS3, .MSn
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Quadrupole-q-TOF
ESI QqTOF
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installation phase.
FT MS
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bottom line
..Resolution and mass accuracy
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FWHM

• Full width at half maxima of a peak

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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
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Mass accuracy is measured as parts per million
value
ppm 106?m 106 M
R
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outline

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

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Peptide Mass Fingerprinting - PMF
Database entry NCBI
From http//gobi.ym.edu.tw/course/mass/2004-0325.
pdf
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Informatics

• Search engines
• Mascot, Matrix Science
• Sequest, Thermoelectron
• Free-ware
• Protein prospector (http//prospector.ucsf.edu/)
• TPP tools (http//tools.proteomecenter.org/TPP.php
  )

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Database searching using MASCOT Overview of the
experiment Submission of data to MASCOT
webserver
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1D SDS PAGE of proteins
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
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Mass spectrum
Intensity
Mass to charge ratio (m/z)
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Peak list

• Compiled from the mass spectra
• Mass list
• Mass list and intensity
• Submitted to the search engine

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http//www.matrixscience.com/
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Mascot scoring
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List of common contaminants

• Trypsin autolysis peptides
• Matrix peaks
• Keratin from skin, hair
• Other contaminants

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Protein Identification
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
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Tandem mass spectrum
http//qbab.aber.ac.uk
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Tandem mass spectrum
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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
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(No Transcript)
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Top hits from Mascot Search there are multiple
accession numbers for the same protein
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Search returns a cluster of proteins with the
same matching peptides
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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
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outline

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

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Quantitative Proteomics
Sample preparation
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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
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..its high-throughput
1st Dimension - Isoelectric focussing
2nd Dimension SDS PAGE
Spot picking
Trypsin gel digest
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Colorectal cancer markers
Balasubramani et al., Cancer Res., 2006
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Shotgun proteomics
Adapted from Aebersold, R. Mann, M. Nature 2003,
422, 198-207
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typical workflow to identify biomarkers that
distinguish indolent versus aggressive forms of
cancer..
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Sample handling
In-solution Isoelectric focussing
1D or 2D LC MALDI
HPLC
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Protein-protein interaction studies

• Immunoaffinity pull-downs
• Tandem affinity purification

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GPCL

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

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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

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Isotopic species of M

• (M H) (M 1H)/1H
• (M 2H)2 (M 2H)/2H
• (M 3H)3 (M 3H)/3H