Peptide Mass Fingerprinting for Protein identification

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Peptide Mass Fingerprinting
Manimalha Balasubramani Genomics and Proteomics
Core Laboratories
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Genomics and Proteomics Core Lab
websitewww.genetics.pitt.edu
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GPCL Inventory

• ABI Voyager DE PRO, user operated
• 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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4700 Proteomics Analyzer, ABI
Voyager DE PRO, ABI
micrOTOF, Bruker
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LCQ Deca XP, Thermofisher
12T FT MS, Bruker
Qstar Elite, ABI
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Peptide mass fingerprinting (PMF) is a technique
for protein and peptide identification
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Outline

• PMF Workflow
• Sample preparation
• Mass spectra MS, and MS/MS
• Database searches
• Examples, hands-on exercises
• Contaminants, post-translational modifications,
  enzyme digestions
• Evaluating PMF analysis

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PMF Sample preparation
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Mass Spectra are acquired with..

• MALDI TOF MS (Voyager DE PRO, ABI)
• MALDI TOF/TOF MS (4700 Proteomics Analyzer, ABI)
• MALDI Matrix Assisted Laser Desorption
  Ionization
• TOF Time Of Flight
• MS Mass Spectrometry

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Mass Spectrum MS
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FWHM
Full width at half maxima of a peak
Source wiki
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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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Ubiquitin ESI Spectra on 12T FT-ICRMass Error gt
0.56 ppm
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Ubiquitin ESI Spectra on 12T FT-ICRMass Error lt
0.56 ppm
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Ubiquitin ESI Spectra 12T FT-ICR Resolution gt
175,000
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Mass accuracy is measured as parts per million
value
ppm 106?m 106 M
R
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Peptide Mass Fingerprint
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Mass spectrum processing, calibration

• External calibration
• Internal calibration
• trypsin autodigestion peaks
• Keratin peaks
• Spiking with an internal standard

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Peak List

• Spectrum viewer
• Compiled from the mass spectra
• Mass list
• Mass list and intensity
• Peak list is submitted for Database searching

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Database searching
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Description of database searching using Mascot
program

• At GPCL, 4800 Proteomics analyzer data is
  presented to the Mascot webserver through
  ProteinPilot
• Mascot can be accessed through the web
• http//www.matrixscience.com

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Mascot scoring
Source http//www.matrixscience.com/
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PMF search page
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Parameters used in database searching

• Database searched
• Taxonomy
• Enzyme
• Missed cleavages
• Fixed versus variable modifications (PTMs)
• MW and pI
• Mass tolerance

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Oxidation of methionine in proteins and peptides
32 Da
16 Da
From Ionsource.com
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S-carboxymethylation of the amino acid residue
cysteine with the alkylating agent iodoacetic
acid  Or s-carbamidomethylation with
iodoacetamide (57 da)
58 Da
From Ionsource.com
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Databases NCBI
nr.tar.gz non-redundant protein sequence
database with entries from GenPept, Swissprot,
PIR, PDF, PDB, and NCBI RefSeq
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Swiss-Prot, IPI, others
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Submit a peak list to Mascot

• 1075.513062
• 1086.581177
• 1090.547241
• 1092.517822
• 1100.630249
• 1103.572754
• 1106.553223
• 1107.529663
• 1118.498779
• 1119.519531
• 1121.509644
• 1129.604492
• 1141.572388
• 1156.586792
• 1166.537231
• 1170.607422
• 1172.612183
• 1179.590332
• 1194.604126

• 1427.830566
• 1435.718872
• 1475.762695
• 1479.710327
• 1493.734131
• 1502.774780
• 1530.834717
• 1575.850952
• 1607.807007
• 1629.868408
• 1639.935425
• 1752.863892
• 1753.904663
• 1754.915161
• 1791.744507
• 1792.805054
• 1794.820801
• 1816.801392
• 1875.976196

http//matrixscience.com/cgi/search_form.pl?FORMVE
R2SEARCHPMF
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Mascot PMF report
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Hands-on exercise

• Go to Desktop
• open txt file
• copy and paste in Mascot search page
• Specify search parameters
• Allow 100ppm error for PMFal_100.txt
• Allow 25ppm error for PMFgd_25.txt

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Not all peaks are matched why?

• Theoretical peptide list
• peptides lengths vs. MS range
• Enzyme missed/non-specific cleavage
• Incorrect ORF
• Amino acid substitutions
• Ion suppression/efficiency

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Not all peaks are matched why?

• Experimental peptide list
• Contaminants
• Trypsin autolysis peptides
• Hair, skin keratins
• Matrix molecules, clusters
• Unknown contaminants
• Modifications
• PTMs known and unknown, biological origin
• Oxidized methionines, gel induced artifacts
• Chemical cysteine carbamidomethylation, sample
  handling introduced
• Adducts
• Amino acid substitutions
• Splice variant

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Database search takes into account contaminants,
modifications, For eg.
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Evaluating PMF analysis

• Acceptable hit
• High score
• Major peaks accounted for
• No hit
• Insufficient data low intensity MS
• Single gel band contains gt2-3 proteins
• Protein not represented in database ORF/genome
• Further analysis
• MS/MS confirmation of few major peaks,
  unaccounted peaks Ideal
• Low score, good spectrum LC MS/MS
• Low score, low intensity spectrum concentrate
  sample, reacquire
• High score, some unaccounted peaks MS/MS

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MS/MS

• Plot of m/z versus intensity
• At GPCL,
• MALDI TOF/TOF MS
• ESI QqTOF MS
• ESI IT MS
• MALDI/ESI FT ICR MS

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Tandem MS
4700 Proteomics Analyzer, Applied Biosystems
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MS

• MS, followed by precursor ion selection

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Fragment ion spectrum
Tandem MS
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Tandem mass spectrum
http//qbab.aber.ac.uk
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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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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
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GPCL resources for Bioinformatic analysis

• Mascot version 2.1.0, Matrix Science Ltd
• Mascot Daemon
• ProteinPilot software 2.0, Applied Biosystems/MDS
  Sciex
• Paragon algorithm
• And Mascot algorithm
• Sequest, Thermoelectron

Selected list
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Resources

• http//www.hsls.pitt.edu/guides/genetics/obrc/prot
  eomics

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..its high-throughput
1st Dimension - Isoelectric focussing
2nd Dimension SDS PAGE
Spot picking
Trypsin gel digest
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Sample separation..
In-solution Isoelectric focussing
1D or 2D LC MALDI
HPLC
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GPCL services..

• Fee for service model
• Support investigators
• Scientific expertise
• Technical expertise
• Grant submission

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• Genomics and Proteomics Core Laboratories
• Paul Wood Billy W. Day
• Director Scientific Director
• Janette Lamb
• Assistant Director
• Proteomics Lab
• Chris Bolcato
• John Cardamone
• Emanuel M Schreiber
• Guy Ueichi
• James Porter
• Robert Wolfe

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A mass spectrum

• Plot of m/z versus intensity
• MALDI TOF (/TOF) MS
• ESI TOF MS
• ESI QqTOF MS
• ESI IT MS
• MALDI/ESI FT ICR MS

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Mass analyzers several designs
Aebersold and Mann, Nature review, 422, p198, 2003
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QqTOF MS/MS
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Each search engine scores differently
SEQUEST
But the overlap is surprisingly small. Different
search engines match different spectra.
Each search engine identifies about the same
number of spectra,
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22
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Mascot
X!tandem
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Courtesy Proteome Software Inc.
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