Peptide Mass Fingerprinting Manimalha Balasubramani Genomics and Proteomics Core Laboratories

Genomics and Proteomics Core Lab website

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

4700 Proteomics Analyzer, ABI Voyager DE PRO, ABI micrOTOF, Bruker

LCQ Deca XP, Thermofisher 12T FT MS, Bruker Qstar Elite, ABI

Peptide mass fingerprinting (PMF) is a technique for protein and peptide identification

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

PMF: Sample preparation Peptide fingerprint

Mass Spectra are acquired with.. MALDI TOF MS (Voyager DE PRO, ABI) MALDI TOF/TOF MS (4700 Proteomics Analyzer, ABI) MALDI – M atrix A ssisted L aser Desorption I onization TOF – T ime O f F light MS – M ass S pectrometry

Mass Spectrum: MS Mass to charge ratio (m/z) Intensity

FWHM Full width at half maxima of a peak Source: wiki

Resolution and mass accuracy R = M Δm R = resolution M = mass of the peak of interest Δ m = width in daltons of the peak Δm measured at 50% peak height is the Full Width at Half Maxima (FWHM)

Ubiquitin ESI Spectra on 12T FT-ICR Mass Error > 0.56 ppm

Ubiquitin ESI Spectra on 12T FT-ICR Mass Error < 0.56 ppm

Ubiquitin ESI Spectra 12T FT-ICR Resolution > 175,000

Mass accuracy is measured as parts per million value ppm = 10 6 Δm = 10 6 M R

Peptide Mass Fingerprint

Mass spectrum processing, calibration External calibration Internal calibration –trypsin autodigestion peaks –Keratin peaks –Spiking with an internal standard

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

Database searching

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 -

Mascot scoring A frequency factor matrix, F, is created, in which each row represents an interval of 100 Da in peptide mass, and each column an interval of 10 kDa in intact protein mass. As each sequence entry is processed, the appropriate matrix elements fi,j are incremented so as to accumulate statistics on the size distribution of peptide masses as a function of protein mass. The elements of F are then normalised by dividing the elements of each 10 kDa column by the largest value in that column to give the Mowse factor matrix M: After searching the experimental mass values against a calculated peptide mass database, the score for each entry is calculated according to: Where MProt is the molecular weight of the entry and the product term is calculated from the Mowse factor elements for each match between the experimental data and peptide masses calculated from the entry. Source:

PMF search page

Parameters used in database searching Database searched Taxonomy Enzyme Missed cleavages Fixed versus variable modifications (PTMs) MW and pI Mass tolerance

Oxidation of methionine in proteins and peptides +16 Da +32 Da From Ionsource.com

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

Databases: NCBI nr.*tar.gz non-redundant protein sequence database with entries from GenPept, Swissprot, PIR, PDF, PDB, and NCBI RefSeq

Swiss-Prot, IPI, others

Submit a peak list to Mascot

Mascot PMF report

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

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

Experimental peptide list –Contaminants Trypsin autolysis peptides Hair, skin keratins Matrix molecules, clusters Unknown contaminants –Modifications PTM’s – known and unknown, biological origin Oxidized methionines, – gel induced artifactsOxidized methionines, Chemical – cysteine carbamidomethylation, sample handling introducedcysteine carbamidomethylation Adducts Amino acid substitutions Splice variant Not all peaks are matched –why?

Database search takes into account contaminants, modifications, For eg.

Evaluating PMF analysis Acceptable hit –High score –Major peaks accounted for No hit –Insufficient data – low intensity MS –Single gel band contains >2-3 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

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

Tandem MS 4700 Proteomics Analyzer, Applied Biosystems

MS MS, followed by precursor ion selection

Fragment ion spectrum Tandem MS

Tandem mass spectrum

Database Searching Peptide Mass Fingerprinting Sequence tag approach De novo sequencing inspect raw data Tandem mass spectra (MS/MS) can be used for peptide sequencing

Mascot Search Results Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID: 15790, SpectrumID: 17225, Path=\Mani\102004\New Analysis 1 Database : NCBInr ( sequences; residues) Timestamp : 20 Oct 2004 at 14:52:50 GMT Top Score : 681 forgi|180570, creatine kinase [Homo sapiens] Probability Based Mowse Score Score is-10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 75 are significant (p<0.05).

Top hits from Mascot Search – there are multiple accession numbers for the same protein

Search returns a cluster of proteins with the same matching peptides

Nominal mass (M r ): 42591; Calculated pI value: 5.34 Observed Mass & pI: 43kd, Creatine kinase - B [Homo sapiens] Match to: gi| ; Score: 681 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 Creatine kinase B is the highest scoring protein

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

Resources /proteomics

2 nd Dimension – SDS PAGE 1 st Dimension - Isoelectric focussing Spot picking Trypsin gel digest..its high-throughput…

Sample separation.. HPLC 1D or 2D LC MALDI In-solution Isoelectric focussing

GPCL services.. Fee for service model Support investigators –Scientific expertise –Technical expertise –Grant submission

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

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

Mass analyzers – several designs Aebersold and Mann, Nature review, 422, p198, 2003

QqTOF MS/MS

9% 19%7% 34% 5% 4%22% Mascot Each search engine identifies about the same number of spectra, But the overlap is surprisingly small. Different search engines match different spectra. But the overlap is surprisingly small. Different search engines match different spectra. Each search engine scores differently SEQUEST X!tandem Courtesy: Proteome Software Inc.

James Lyons-Weiler Scientific Director Bioinformatics Analysis Core (412) (office) (412) (cell) Fax: