Shotgun Proteomic Analysis

Department of Cell BiologyThe Scripps Research Institute

Biological/Functional Resolution of Experiments

Organelle

Cells/Tissues

Multiprotein Complex

Function

InteractionAnalysis

ExpressionAnalysis

InformationContent

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µLC

“Shotgun Proteomics”

…SEQ

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T80 node Beowulf Computer

Cluster

Protein Mixture

proteolysis

Peptide Mixture

Output Filtering and Re-Assembly

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Data Processing Issues with Shotgun Proteomics

1:1 Mixture of Unlabeled/15N-Labeled YeastSoluble Proteins Analyzed Using a Single 12h Analysis

304 (1.9x)157Protein ID’s(*2 peptides confirmed w/ RelEx)

891 (1.6x)559Protein ID’s (*1 peptide confirmed w/ RelEx)

86,950 (4.5 x)18,970MS/MS SpectraLTQLCQ

*RelEx was used to evaluate the presence of labeled isotopomer

Processing Tandem Mass SpectraProcessing Tandem Mass Spectra

Spectral Quality

Database Analysis

De Novo: Unusual,Unanticipated Features

Filter

SEQUEST™PepProb

GutenTag

Quantification RelX

Subtractive Analysis LibQUESTDTASelect/Contrast

Filter and Assembly DTASelectProtProb

Data Issues

• Data quality• How is a match determined?

– Protease issues?– Validation issues?

• Posttranslational Modifications?• Quantification?• Sampling Issues?

Spectral Filtering with Hand Crafted FeaturesSpectral Filtering with Hand Crafted Features

Called Good Called Bad %Correct+1 GOOD 671 75 89.9%+1 BAD 5585 11475 67.3%+2/+3 GOOD 3166 348 90.1%+2/+3 BAD 11611 26684 69.7%All GOOD 3837 423 90.1%All BAD 17196 38159 68.9%

Bern, Goldberg, MacDonald, Yates Bioinformatics (in press)

•• Identification of Identification of PTMsPTMs

•• Sample is split into 3 aliquotsSample is split into 3 aliquots

•• Digest using 3 different proteasesDigest using 3 different proteases

•• Mix and analyze by LC/LCMix and analyze by LC/LC--MS/MSMS/MS

•• Interpret spectra using SEQUESTInterpret spectra using SEQUEST

MudPIT

SEQUEST

trypsin elastase subtilisin

MultiMulti--Enzyme Digestion ProceduresEnzyme Digestion Procedures

High pH

Proteinase K

High pH/Proteinase K Method(hpPK Method)

Wu et al., Nat. Biotech. 21:532-538 (2003)Howell and Palade, J. Cell Biol. 92:822-832 (1982)

Overlapping Peptide Coverage(TM7) gi|13794265|ref|NP_056312.1|DKFZP564G2022 protein MAAAAWLQVL PVILLLLGAH PSPLSFFSAG PATVAAADRS KWHIPIPSGK NYFSFGKILF RNTTIFLKFD GEPCDLSLNI TWYLKSADCY NEIYNFKAEE VELYLEKLKE KRGLSGNIQT SSKLFQNCSE LFKTQTFSGD FMHRLPLLGE KQEAKENGTN LTFIGDKTAM HEPLQTWQDA PYIFIVHIGI SSSKESSKEN SLSNLFTMTV EVKGPYEYLT LEDYPLMIFF MVMCIVYVLF GVLWLAWSAC YWRDLLRIQF WIGAVIFLGM LEKAVFYAEF QNIRYKGESV QGALILAELL SAVKRSLART LVSIVSLGYG IVKPRLGVTL HKVVVAGALY LLFSGMEGVL RVTGAQTDLA SLAFIPLAFL DTALCWWIFI SLTQTMKLLK LRRNIVKLSL YRHFTNTLIL AVAASIVFII WTTMKFRIVT CQSDWRELWV DDAIWRLLFS MILFVIMVLW RPSANNQRFA FSPLSEEEEEDEQKEPMLKE SFEGMKMRST KQEPNGNSKV NKAQEDDLKW VEENVPSSVT DVALPALLDS DEERMITHFE RSKME

WVEENVPSSVTDVALPALLDS*DEERVEENVPSSVTDVALPALLDS*DEER

EENVPSSVTDVALPALLDS*DEERENVPSSVTDVALPALLDS*DEER

VPSSVTDVALPALLDS*DEERPSSVTDVALPALLDS*DEER

LPALLDS*DEERPALLDS*DEER

(TM6) gi|14249524|ref|NP_116213.1| hypothetical protein FLJ14681MVAACRSVAG LLPRRRRCFP ARAPLLRVAL CLLCWTPAAV RAVPELGLWL ETVNDKSGPL IFRKTMFNST DIKLSVKSFH CSGPVKFTIV WHLKYHTCHN EHSNLEELFQ KHKLSVDEDF CHYLKNDNCW TTKNENLDCN SDSQVFPSLN NKELINIRNV SNQERSMDVV ARTQKDGFHI FIVSIKTENT DASWNLNVSL SMIGPHGYIS ASDWPLMIFY MVMCIVYILY GILWLTWSAC YWKDILRIQF WIAAVIFLGM LEKAVFYSEY QNISNTGLST QGLLIFAELI SAIKRTLARL LVIIVSLGYG IVKPRLGTVM HRVIGLGLLY LIFAAVEGVM RVIGGSNHLA VVLDDIILAV IDSIFVWFIF ISLAQTMKTL RLRKNTVKFS LYRHFKNTLIFAVLASIVFM GWTTKTFRIA KCQSDWMERW VDDAFWSFLF SLILIVIMFL WRPSANNQRY AFMPLIDDSD DEIEEFMVTS ENLTEGIKLR ASKSVSNGTA KPATSENFDE DLKWVEENIP SSFTDVALPV LVDSDEEIMT RSEMAEKMFS SEKIM

WVEENIPSSFTDVALPVLVDS*DEEIMTRIPSSFTDVALPVLVDS*DEEIMTRPSSFTDVALPVLVDS*DEEIMTR

SFTDVALPVLVDS*DEEIMTRTDVALPVLVDS*DEEIMTRTDVALPVLVDS*DEEIMTRS

DVALPVLVDS*DEEIMTRVALPVLVDS*DEEIMTR

ALPVLVDS*DEEIMTRALPVLVDS*DEEIMTRS

LPVLVDS*DEEIMTRPVLVDS*DEEIMTRPVLVDS*DEEIMTRS

gi|27229118|ref|NP_082129| RIKEN cDNA 0610006F02; S-adenosylmethionine-dependentmethyltransferase activity [Mus musculus]

MDALVLFLQL LVLLLTLPLH LLALLGCWQP ICKTYFPYFM AMLTARSYKK MESKKRELFS QIKDLKGTSG NVALLELGCG

TGANFQFYPQ GCKVTCVDPN PNFEKFLTKS MAENRHLQYE RFIVAYGENM KQLADSSMDV VVCTLVLCSV QSPRKVLQEVCVDPN PNFEKF

VTCVDPN PNFEKVTCVDPN PNFEKFLTK

QRVLRPGGLL FFWEHVAEPQ GSRAFLWQRV LEPTWKHIGD GCHLTRETWK DIERAQFSEV QLEWQPPPFR WLPVGPHIMGQFSEV QLEWQPPPFR WLPVGPHIM

EV QLEWQPPPFR WLPVGPHEV QLEWQPPPFR WLPVGPHEV QLEWQPPPFR WLPVGPHIMEV QLEWQPPPFR WLPVGPHIM**LEWQPPPFR WLPVGPHLEWQPPPFR WLPVGPHLEWQPPPFR WLPVGPHIMLEWQPPPFR WLPVGPHIMLEWQPPPFR WLPVGPHIMGEWQPPPFR WLPVGPHIMWQPPPFR WLPVGPH

KAVK

Golgi Peptide Synthetic Peptide

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Dimethyl Arginine Containing Peptide

Shotgun Proteomic Experiments Shotgun Proteomic Experiments and Sampling Issuesand Sampling Issues

•• Based on prior studies in yeast, we know Based on prior studies in yeast, we know not every protein present is not every protein present is id’did’d

•• Reproducibility is good for high abundance Reproducibility is good for high abundance proteins 70proteins 70--80%80%

•• Reproducibility is not as good for low Reproducibility is not as good for low abundance proteins. 20abundance proteins. 20--30%30%

•• Is this predictable?Is this predictable?

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Experimental Semi-empirical Model Random Model

Random Sampling Model for Data Dependent Acquisition

))/1(1(* SL NLnK −−=

nL= # of protein species at particular levelL = abundance levelN = total number of proteinsS = experiments

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Distribution of Protein Identifications AfterRepeating Analysis 9 times

# of proteins identifiedin all 9 experiments

# of proteins identifiedin 1 experiment

RelX software

DTASelect OutputPeptide SequenceLVNHFIQEFK

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5) Correlation

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Protein Mixture Ratio

TSA1 = 1.335±0.015

SSA1 = 1.004±0.018

ADH1 = 0.661±0.045

Systematic Errors are Present in Samples

Spectral Sampling for Relative QuantificationSpectral Sampling for Relative QuantificationCombined Data for 6 proteins added to Yeast SolubleCombined Data for 6 proteins added to Yeast Soluble

Cell Lysate at 4 different levelsCell Lysate at 4 different levels

y = 1.0613x + 0.2608R2 = 0.9997

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Linear dynamicrange 2-orders

Measuring small changesis not as reliable

Synthesis of Ribosomal Proteins in Plasmodium falciparum

• Striking trend: almost all ribosomal proteins increase over ring to trophtransition

Ribosomal Protein Abundance

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Standards

• 1. Data formats: McDonald et al MS1, MS2, and SQT - three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications, RCM 2004, 18, 2162-8.

• Database search standard based on: Washburn et al, Large Scale analysis of the yeast proteome via

multidimensional protein identification technology Nature Biotechnology 19, 242-247 (2001)

MacCoss et al , Probability Based Validation of Protein Identifications Using a Modified SEQUEST Algorithm, Analytical Chemistry 74, 5593-5599 (2002). Normalized Scores

Standards

• 4. Standards should not prevent innovation– Data formats should be practical e.g. storage space

• 7. Data processing tools should be transparent and validated, e.g. published

• 8. Data for publication: information to support biological conclusions- sequences of peptides id’d.

• 9. Archiving data: biological conclusions should be the most important part of the experiment

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Probability Distributions

Single Spectral Matches are Problematic: How Single Spectral Matches are Problematic: How to tell if they are correctto tell if they are correct

•• Searches determine closeness of fit based Searches determine closeness of fit based on some measure: Compare matches with on some measure: Compare matches with different programsdifferent programs

•• Probability scoringProbability scoring: P = random match : P = random match based on frequency of fragment ions in based on frequency of fragment ions in databasedatabase

•• SEQUESTSEQUEST: XCorr measures how close the : XCorr measures how close the spectrum fits to ideal spectrumspectrum fits to ideal spectrum

•• Manual validation, experimental validationManual validation, experimental validation•• de novode novo interpretationinterpretation

•• Sample is split into 3 aliquotsSample is split into 3 aliquots

•• Digest using 3 different proteasesDigest using 3 different proteases

trypsin elastase subtilisin

MultiMulti--Enzyme DigestEnzyme Digest

•• Sample is split into 3 aliquotsSample is split into 3 aliquots

•• Digest using 3 different proteasesDigest using 3 different proteases

•• Mix and analyze by LC/LCMix and analyze by LC/LC--MS/MSMS/MS

•• Interpret spectra using SEQUESTInterpret spectra using SEQUESTMudPIT

SEQUEST

trypsin elastase subtilisin

MultiMulti--Enzyme DigestEnzyme Digest

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% of Prote insIdentified

% of UniquePeptides

% of SpectrumCopies

P1P2P3P4

Properties of Data Dependent Data Acquisition

Most invariant property is spectral copy number

GutenTagGutenTag: Partial de novo Sequencing : Partial de novo Sequencing of Tandem Mass Spectraof Tandem Mass Spectra

•• Database searching assumes minimal errors in the Database searching assumes minimal errors in the database and sequence variations between strains, database and sequence variations between strains, individuals and speciesindividuals and species

•• Modifications need to be specified in database Modifications need to be specified in database searches, so unanticipated modifications will be searches, so unanticipated modifications will be missed.missed.

•• Partial Partial De novoDe novo analysis of tandem mass spectra in analysis of tandem mass spectra in largelarge--scalescale can identify peptides containing can identify peptides containing sequence variations and unanticipated sequence variations and unanticipated modifications modifications

GutenTagGutenTag

•• 6170 tandem mass spectra: LC/LC/MS/MS 6170 tandem mass spectra: LC/LC/MS/MS analysis of simple digested protein mixtureanalysis of simple digested protein mixture

•• 1987 spectra matched by SEQUEST1987 spectra matched by SEQUEST•• 1328 spectra matched by GutenTag1328 spectra matched by GutenTag•• 766 partial matches suggesting modifications and 766 partial matches suggesting modifications and

sequence variationssequence variations•• Total matching spectra by Total matching spectra by GutenTagGutenTag: 2,094: 2,094•• Partial de novo will extend identificationsPartial de novo will extend identifications•• Software is Software is AutomatedAutomated and and LargeLarge--ScaleScale

Improved Spectral Quality Effects Peptide IdentificationInfusion of 1 pmol/µl Angiotensin I

LCQ-Classic761 of 1000 MS/MS Spectra Matched the Correct Sequence

LTQ970 of 1000 MS/MS Spectra Matched the Correct Sequence

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Database Searching with Tandem Database Searching with Tandem Mass SpectraMass Spectra

•• The goal is to identify peptides using MS/MS The goal is to identify peptides using MS/MS spectra and amino acid sequence databases.spectra and amino acid sequence databases.

•• Develop a probabilistic model that establishes a Develop a probabilistic model that establishes a relationship between the database sequences and relationship between the database sequences and the spectrum to complement quantitative measures the spectrum to complement quantitative measures of closenessof closeness--ofof--fit fit

•• Develop nonDevelop non--empirical probabilistic measures empirical probabilistic measures using crossusing cross--correlation measurementscorrelation measurements

Probability Model

• Null hypothesis: All fragment matches to MS/MS spectrum are by random.

• N, K – number of all fragments and all fragment matches, respectively. N1 is the number of fragments of a particular peptide which has K1 matches.

• We seek an amino acid sequence that has the smallest probability of being a random match.

1

111 *),( 11, NN

KNKN

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NK CCCNKP

−−=

Model Hypergeometric Distributions

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Number of Successes

N, K – number of all fragments and all fragment matches, respectively. N1 is the number of fragments of a particular peptide which has K1 matches.

Significance of Peptide IdentificationSignificance of Peptide Identification

•• Not all identifications are significant: poor quality Not all identifications are significant: poor quality spectra of peptides , incomplete peptide spectra of peptides , incomplete peptide fragmentation, inaccuracies in database, fragmentation, inaccuracies in database, posttranslational modifications, MS/MS of posttranslational modifications, MS/MS of chemical noise and nonchemical noise and non--peptide molecules.peptide molecules.

•• Significance of a match (P_value) is also obtained Significance of a match (P_value) is also obtained from the hypergeometric distribution.from the hypergeometric distribution.

Significance of a Match

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Yeast Database

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N=5284150K=569160N1=40

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Number of Fragment Matches

Cumulative Distribution

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Mass/Charge State Dependence

• Scores that use closeness of fit measures can artificially inflate with weight/mass.

• This complicates use of uniform criteria for identification.

• Probabilities generated by hypergeometric distribution are charge/weight independent.

Cross-Correlation Score Distribution

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Cross-Correlation Scores

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Database Dependence

• The probability inferred from the hypergeometric distribution is in principle database dependent.

• However, the dependency is very weak.

NRP and Yeast Databases

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Pep_Probe SummaryPep_Probe Summary

•• Implements 4 scoring schemes: hypergeometric, Implements 4 scoring schemes: hypergeometric, poissonpoisson, , maximum likelihood and crossmaximum likelihood and cross--correlation. Sorts results correlation. Sorts results either by hypergeometric or crosseither by hypergeometric or cross--correlation scores.correlation scores.

•• No enzyme specificity is assumed.No enzyme specificity is assumed.•• Reports significance of each match.Reports significance of each match.•• Can search for posttranslational modifications to three Can search for posttranslational modifications to three

different amino acids.different amino acids.•• Has been implemented to run on a standalone or compute Has been implemented to run on a standalone or compute

clusters.clusters.•• Runs on heterogeneous cluster of computers, in WINDOWS Runs on heterogeneous cluster of computers, in WINDOWS

and LINUX platforms.and LINUX platforms.

Processing Tandem Mass SpectraSpectral Quality

Database Analysis

Analyze Unusual orUnanticipated Features

Eliminate Poor Quality Spectra, Score Quality of

Other spectra

Multiple Methods With Different

Selectivity's

De novo or partialDe novo analysis

Assemble and Annotate Data Biological Significance

Increased Data Production Requires Automated Data AnalysisIncreased Data Production Requires Automated Data Analysis

MS/MS MS/MS ComparisonComparison

Library SearchingLibrary SearchingComparative AnalysisComparative AnalysisSubtractive AnalysisSubtractive Analysis

LIBQUEST LIBQUEST

Yates et al. Yates et al. Anal.Chem. Anal.Chem. 70, 3557 (1998)70, 3557 (1998)

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De NovoDe NovoSequencingSequencing

Alternate SplicingAlternate SplicingUnanticUnantic. Mod.. Mod.Unknown ORFsUnknown ORFs

GutenTagGutenTag

Tabb et al Anal. Tabb et al Anal. ChemChem

Existing SequenceExisting SequencePTM PTM

Database Search

Database Database SearchSearch

SEQUEST & Pep_ProbSEQUEST & Pep_ProbYates et al.,Yates et al., JASMS JASMS 5, 976 (1994)5, 976 (1994)Yates et al. Yates et al. Anal. Anal. ChemChem 67, 1426 (1995)67, 1426 (1995)Yates et al. Yates et al. Anal. ChemAnal. Chem. 67, 3205 (1995). 67, 3205 (1995)MacCoss et al. MacCoss et al. Anal. Anal. ChemChem (2002)(2002)Sadygov and Yates, Sadygov and Yates, Anal. Anal. ChemChem (in press)(in press)

DTASelectDTASelectPost Analysis Post Analysis

Review SoftwareReview Software

Link et al. Link et al. Nature Biotech. Nature Biotech. 1717, 676, 676--682 (1999)682 (1999)Tabb et. al. Tabb et. al. J. Proteome Res.J. Proteome Res. 1, 26, (2002) 1, 26, (2002)

Probability forProbability forProtein Protein

IdentificationIdentification

Link et al. Link et al. Nature Biotech. Nature Biotech. 1717, 676, 676--682 (1999)682 (1999)Tabb et. al. Tabb et. al. J. Proteome Res.J. Proteome Res. 1, 26, (2002) 1, 26, (2002)

Peptide IdPeptide IdProbabilityProbabilityPP--Val, S.C.Val, S.C.

Related SequenceRelated SequenceSNP AnalysisSNP AnalysisMutation AnalysisMutation Analysis

VariantVariantSearchSearch

SEQUESTSEQUEST--SNPSNP

Gatlin et al.Gatlin et al. Anal.Chem.Anal.Chem. 72, 757 (2000). 72, 757 (2000).

SpectralQuality

Bioinformatics (in press)Bioinformatics (in press)

Data Considerations

• Different types of experiments• Different types of data analysis

Integrated Multi-Dimensional Liquid Chromatography

hV

100 micron FSC

RPSCXRP

5 µM100-300 nL/min

WasteWaste

Comprehensive Analysis of Complex Protein Mixtures

Total ProteinCharacterization

• Protein Identification: What’s there• Post Translational Modifications: Regulation• Quantification: Dynamics• Proteomic Data to Knowledge: Genetics, RNAi, siRNA

Multiprotein Complex/OrganelleCells/Tissues

Translation of technology development into biological discoveryTranslation of technology development into biological discovery