GenomicPeptideFinder:
Mass spectrometric data mining in genomic data bases
Institute for Plant Biochemistry and Biotechnology University of Münster, Germany
Peng, J. and Gygi, S.P. (2001) Proteomics: the move to mixtures. J. Mass Spectrom., 36,
1083-1091.
Schematic depiction of an ion trap mass spectrometer
Mass Spectra Recording (Triple Play)
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Fragmentation Spectrum
M /Z
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nsity
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4 0 0 x 1 0 3
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SLSPAQVSFAVEAK G V
KVAEVAFSVQAPS L S
b 1 4b 1 3b 1 2b 1 1b 1 0b 9b 8b 7b 6b 5b 4b 3
y 1 5y 1 4y 1 3y 1 2y 1 1y 1 0y 9y 8y 7y 6y 5y 4y 3
G
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H2N—CH—C—NH—CH—C—NH—CH—C—NH—CH—C—OH
R1 R3R2 R4
O OO O
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zsslifi
Correlation analysis
# deltCn XCorr Sp Reference Peptide1. 0.0000 4.2902 382.4 1152RZ (-)GSGDAAYPGGPFFNLFNLGK2. 0.5359 1.3963 70.0 582RZ (V)VCLMSAIALPYNGGLRPPGV3. 0.6028 1.1949 43.1 258RZ (-)GGGVVCFDASFRSPKFGIGK5. 0.6160 1.1553 70.1 2360RZ (-)VDSGWGGVVVVALAPYNLGR6. 0.6666 1.0029 37824 37RZ (K)CVLWAYPGVALGRVQVLGK11. 0.7333 0.8022 37647 304RZ (V)GGWLGRLVGGATVLGKAYPGV12. 0.7351 0.7970 29.2 1080RZ (-)IAAYPGVSPGLMIHYNIGR13. 0.7774 0.6695 22.0 304RZ (K)AYPGVGELVRGGCMMCSIGK14. 0.7842 0.6492 31.4 37RZ (W)AYPGVALGRGIRLGKCVLW
Database selection
>1080ZRIAAYPGVSPGLMIHYNIGR>1137RZAAYPGATQPGATELARRLGK>1152RZGSGDAAYPGGPFFNLFNLGK>1152ZRGSGDAAYPGGPFFNLFNLGK>2360RZVDSGWGGVVVVALAPYNLGR>240RZHPGVVCRPGRGGGCSRHIGKHPGVVCCSRHRRSHTIGK
Problem
• Well resolved mass spectra without identification after database search
• De novo amino acid sequencing with subsequent manual search lead to peptide identification
Sequence availability• Every fourth peptide escapes detection by MS due to an intron which
splits the peptide on the “genomic level”– Choudhary, J. S., Blackstock, W. P., Creasy, D. M. and Cottrell, J. S. (2001) Interrogating the human genome using
uninterpreted mass spectrometry data.Trends Biotechnol, 19, S17-22.
• Each human and Chlamy gene statistically contains 8 exons– Deutsch, M. and Long, M. (1999) Intron-exon structures of eukaryotic model organisms. Nucleic Acids Res, 27, 3219-3228.
Merchant, S. et al., (2007) Science; in press
• 40-60% of human genes are alternatively spliced– Modrek, B. and Lee, C. (2002) A genomic view of alternative splicing. Nat Genet, 30, 13-19.
• 50% of a genome may not be complementable by EST data– Seki, M., Narusaka, M., Kamiya, A., Ishida, et. al. (2002) Functional annotation of a full-length Arabidopsis cDNA collection.
Science, 296, 141-145.
• Gene prediction is far from being correct (Only 15-20% of known gene structures are predicted correctly.)
– Brent, M.R. and Guigo, R. (2004) Recent advances in gene structure prediction. Curr Opin Struct Biol, 14, 264-272.
• Post translational modifications– Perkins, D. N., Pappin, D. J., Creasy, D. M. and Cottrell, J. S. (1999) Probability-based protein identification by searching
sequence databases using mass spectrometric data. Electrophoresis, 20, 3551-67.
intron
Right Peptide FragmentLeft Peptide Fragment
Intron
GGPFFNLFNLGK......(K)GSGDAAYP
GTGAGCAGATAGAGGAGAGAGCGCGCGAGAGAGGGCCGCGGCTGCAGCTGGTGTGGGCCAAGGCTGGCGGAGACAGCCAGCAGGGGTCAGGGGGAGGGGCACAGGGCAGAGGCAGACGCCGGCGCTGTCGGGTGCGGGATGCGGGTTGCTGATGGATGGAAACTCCGATCCGCGGCGGACGGTTTCGTGCTATGTAGCTGCTCAACAGGGTTTGCATGCTCCTGGCTGACAGCGCATATGACGGTCCCTTCCCCCGCTCCGCAG
exonintron
Found result
Display resultDeNovo-Sequencingof MS/MS data
RunGenomicPeptideFinder
Database search Found result
Select genomicdatabase
Set optionsRun query
Yes
Yes
MS/MSData
No
Selectsquencedatabase
Verification bydatabase search
0
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180 380 580 780 980 1180
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KA I A Q L E E D Y L
LY D E E L Q A I A K
De Novo Sequencing
1016.4901.6114.8 ~ D (115.02)D
DE
901.6772.4129.2 ~ E (129.1)E
nsi78_11.1803.1806.2.dtaSequence Absolute Relative
Probability Probability[LW]QYSEVLH[AR] 6.4% 41%[PD]SQYSQVLH[AR] 11.5% 31.6%WLQYSEVLH[AR] 4.3% 7.2%
… … …
De Novo Amino Acid Sequence Predictions
De novo amino acidsequencing
Foundresult
Select databaseEnter querySet optionsRun GPF
MS/MSdata
RunGenomicPeptideFinder
Search for sequences for which the mass of the
precursor ion matches the mass of thetryptic peptide.
Store significantcandidates in theresult database
Foundresult
As process 2 butallow for sequenceerrors within the
database.
Foundresult
Combine sequences andcut out putative introns.compare the mass of
the resulting tryptic peptideto the mass of the
precursor ion.
Foundresult
As process 2 butallow for errors in thequery correct these
with the sequences fromthe database.
Process 1 Process 2 Process 3 Process 4
Yes Yes Yes Yes
First (stringent)sequence comparison
Second (less stringent)sequence comparison
RF1: PDPRCHQHQVVRERRNPPRRLVQRLLG-PLHHLLRGDRGYAVR-AAPPSGAGREGQALVGRF2: SIFRHPKQDFGFDPLGLLDPVNSGGFIEPKWLQYSEVIHARWAMLGAAGCIAPEVLGAAGRF3: -SPMPPTSSGSRAA-SPPPARTTATGLTPTPSSSWRSWLCSSLSCAAFRCGEGGAGARRARF4: PLLAVSASLGPHQLQPRPSLARSLLYLLTGVGSVARALEDRLQAQEVLLAHGAGVAEVLQRF5: PCWLSPPALAHTSCSRGPLSRALSSICSPG-AASPEPLKIASRPRKYCWPMEPG-RKSCRRF6: THARRGSVIGCNCNQPQLQRCTRGFK-NRFLRRFFFFARRAPAPPSPHLKAAQLSELHSH
WLQYSEVIHAR 1402.4 Da nsi_63_1.1641.1644.2WLQYS
WLQYS RK EVIHA
Measured Mass: 1402.40 Da
Calculated Mass: 1401.58 Da
Mass Difference: 0.82 Da < 700 ppm (0.98 Da)
De novo amino acidsequencing
Foundresult
Select databaseEnter querySet optionsRun GPF
MS/MSdata
RunGenomicPeptideFinder
Search for sequences for which the mass of the
precursor ion matches the mass of thetryptic peptide.
Store significantcandidates in theresult database
Foundresult
As process 2 butallow for sequenceerrors within the
database.
Foundresult
Combine sequences andcut out putative introns.compare the mass of
the resulting tryptic peptideto the mass of the
precursor ion.
Foundresult
As process 2 butallow for errors in thequery correct these
with the sequences fromthe database.
Process 1 Process 2 Process 3 Process 4
Yes Yes Yes Yes
First (stringent)sequence comparison
Second (less stringent)sequence comparison
RF1: DFRYPGSMGQQYFLGLEAIFKGSGDAAYPGEQIEERARERGPRLQLVWAKAGGDSQQGSGGGAQGRGRRRRCRVRDAGC_RF2: TSATPAPWASSTSWAWRRSSRALATLPTPVSR_RRERAREGRGCSWCGPRLAETASRGQGEGHRAEADAGAVGCGMRVADRF3: LPLPRLHGPAVLPGPGGDLQGLWRRCLPR_ADRGESARERAAAAAGVGQGWRRQPAGVRGRGTGQRQTPALSGAGCGLLRF4: QAAVLDLLELQLLHGSLGLAQVEQVEEGAACGAGEGTVICAVSQEHANPVEQLHSTKPSAADRSFHPSATRIPHPTAPASASRF5: RRPFLISLSFSSFMAASVLPRLNRLKKGPPAERGKGPSYALSARSMQTLLSSYIARNRPPRIGVSIHQQPASRTRQRRRLPLPCARF6: GGRS_SP_ASAPSWQPRSCPG_TG_RRGRLRSGGRDRHMRCQPGACKPC_AAT_HETVRRGSEFPSISNPHPAPDSAGVCLCPV
RF1: WMETPIRGGRFRAM_LLNRVCMLLADSAYDGPFPRSAGGPFFNLFNLGKTEAAMKELKLKEIKNGRLRF2: GWKLRSAADGFVLCSCSTGFACSWLTAHMTVPSPAPQAAPSSTCSTWARPRLP_RS_SSRRSRTAARF3: MDGNSDPRRTVSCYVAAQQGLHAPG_QRI_RSLPPLRRRPLLQPVQPGQDRGCHEGAEAQGDQERPPRF4: ALCPSP_PLLAVSASLGPHQLQPRPSLARSLLYLLTGVGSVARALEDRLQAQEVLLAHGAGVAEVRF5: PLPLTPAGCLRQPWPTPAAAAALSRALSPLSAHRGRQRRQSP_RSPPGPGSTAGPWSRGSGSRF6: DLRVLQPLGLDEAAAVHGVQQAQGVEAEVLFGMTKNGQ-IALMLDRGGMSGHADFIAERL
[RC*] AAYPG[VV]CFNPYNLGK 2027.95 nsi78_03.2460.2463.2
Measured Mass: 2027.95 Da
Calculated Mass: 2027.98 Da
Mass Difference: 0.03 Da < 700 ppm (~ 1.4 Da)
AAYPG
AAYPG RKGSGD EQIEER
GSGDAAYPGGPFFNLFNLGKEQIEER
GSGD EQIEER
Measured Mass ≠ Calculated MassNLGK
NLGK
GPFFNLF
Measured Mass ~ Calculated Mass
Found result
Display resultDeNovo-Sequencingof MS/MS data
RunGenomicPeptideFinder
Database search Found result
Select genomicdatabase
Set optionsRun query
Yes
Yes
MS/MSData
No
Selectsquencedatabase
Verification bydatabase search
MS/MS Data
4 * 1D Gels200 bands
Spectra extraction400 000 spectra
Extract significantResults
Run Sequest(database search)
8 databases
Database
Analysis Workflow: Analysis of thylakoidmembranes after 1D-SDS-PAGE
Allmer et al. (2006) Proteomics 6, 6207-6220.
Database
MS/MSData
Create dta filesfrom raw files2000 dta files
(3 min)
Extract significantResults(1 min)
Run Sequestseveral dbs
Variabel 1 min – 10 h(2h)
PEAKS15 dta files/ min
(2 h)
Create Queriesfor GPF~2000 (3 s)
Lutefisk
DeNovoX
GPFWindows local200 queries / h
(10 h)
GPFUNIX (LINIAC)
60 queries / P*h(3h @ 10 Processors)
Run Sequest on GPF-predictions21 dta files / min
(100 min)
Extract significantResults(1 min)
Total Processing about 14h per raw-file.
AutoMS
LC
MS/MS RawFile
MS Access
Database
PEAKS
Query Creation
GPF
Sequest
Result Extraction
Sequest
Dta-File Setup
Filters Result Extraction
Identified Peptides
47 (4)Complement
105 (17)60 (17)72 (5)PEAKS60 (17)448 (98)322 (19)GPF72 (5)322 (19)2496(22)Sequest
ComplementPEAKSGPFSequest
Confident peptide identifications and complementarities
Number of identified peptides (non redundant) is 2622
411292Comple-ment
1558332GPF
4711571317Sequest
%-missedmissedFound in gene modelsPeptides
Complement PEAKS-Sequest: 3%Improved by using GPF : 18%
Allmer et al. (2006) Proteomics 6, 6207-6220.
Peptides
ALE-GEIYDTFK, bnTY3_06302004.1322.1322.2.dta (Xcorr = 4.1)MEGEFTADNVAK, bnTY3_06302004.920.922.2.dta (Xcorr = 4.12)TGPPAVTVEDADK, bnTY3_06302004.742.746.2.dta (Xcorr = 3.26)TPIVFEGGDR, bnTY3_06302004.1003.1003.2.dta (Xcorr = 2.84)
Gene model: C_390061 [chlre2:164450]
MNRWNLLALTLGLLLVAAPFTKHQFAHASDEYEDDEEDDAPAAPKDDDVDVTVVTVKNWDETVKKSKFALVEFYAPWCGHCKTLKPEYAKAATALKAAAPDALIAKVDATQEESLAQKFGVQGYPTLKWFVDGELASDYNGPRD
ADGIVGWVKKKTGPPAVTVEDADKLKSLEADAEVVVVGYFKALEGEIYDTFKSYAAKTEDVVFVQTTSADVAKAAGLDAVDTVSVVKNFAGEDRATAVLATDIDTDSLTAFVKSEKMPPTIEFNQKNSDKIFNSGINKQLILWTTADDLKADAEIMTVFREASKKFKGQLVFVTVNNEGDGADPVTNFFGLKGATSPVLLGFFMEKNKKFRMEGEFTADNVAKFAESVVDGTAQAVLKSEAIPEDPYEDGVYKIVGKTVESVVLDETKDVLLEVYAPWCGHCKKLEPIYKKLAKRFKKVDSVIIAKMDGTENEHPEIEVKGFPTILFYPAGSDRTPIVFEGGDRSLKSLTKFIKTNAKIPYELPKKGSDGDEGTSDDKDKPASDKDEL* JGI annotation: Protein disulfide isomerase 1 (CrPDI1), encodes the chloroplastic Rb60 protein which is part of a complex involved in the control of psbA mRNA translation by light
NCBI: 0gi|4104541|gb|AAD02069.1|protein disulfide isomerase
SIB-BLAST: 0_CHLRE Protein disulfide isomerase RB60 (EC 5.3.4.1)
Kazusa: 0AF036939.1 Chlamydomonasreinhardtii protein disulfide isomerase
Other:
Scores
Example 1 – Gene model verification
Intron split and parts of intron split peptides are in red: ALE-GEIYDTFKSignificant non-intron split peptides are in blue: MEGEFTADNVAK
1
Scaff: 177403 SDGIVGWVKKKTGPPAVTVEDADKLKSLEADAEVVVVGYFKALE 177272+DGIVGWVKKKTGPPAVTVEDADKLKSLEADAEVVVVGYFKALE
sbjct: 145 ADGIVGWVKKKTGPPAVTVEDADKLKSLEADAEVVVVGYFKALE 188
Scaff: 176994 QGEIYDTFKSYAAKTEDVVFVQTTSADVAKAAGLDAVDTVSVVKNFAGE 176848+GEIYDTFKSYAAKTEDVVFVQTTSADVAKAAGLDAVDTVSVVKNFAGE
sbjct: 188 EGEIYDTFKSYAAKTEDVVFVQTTSADVAKAAGLDAVDTVSVVKNFAGE 236
JGI: tblastnblast protein
vs.translated nucleotide
Peptides
LEEGE-MPLNTYSNK, bnTY5a_062904.1079.1083.2.dta (Xcorr = 4.17)LYDEEL-QAIAK, bnTY5a_062904.1218.1222.2.dta (Xcorr = 4.14)
Gene model: C_1410005[chlre2:155574]
MQTVRAPAASGVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEVSACLGATTGRRKMGRGGERRSGSVGQELAGRILLICDFLFFILQMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPPGTKINSKGKEVPHGTRLYSIASSRYGDDFDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATGTGIAPFRSFWRRCFIENVPSYKFTGLFWLFMGVA
NSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQDMLERGLERNSGESSSEELLLSRTRQGGAGGGGTRTRRAAGRAGGMPRRKAKAPRAGRGTSMSGAV*
JGI annotation: Ferredoxin--NADP reductase, chloroplast precursor. The model doesn't completely fit the published cDNA sequence gi|1706781|sp|P53991|FENR_CHLRE[1706781],
NCBI: 9e-162gi|732532|gb|AAB40978.1|ferredoxin-NADP+ reductase
SIB-BLAST: 1e-162_VOLCA Ferredoxin-NADP+ reductase [fnr] [Volvox carteri]
Kazusa: 1e-170U10545.1 Chlamydomonasreinhardtii ferredoxin-NADP+ reductase
Other:
Scores
Example 2 - Gene model correction
cDNA sequence: FNR cDNA /FENR_CHLRE
MASLRKPSNHADRACSRRLRVATRVAGRRMCRPVAATKASTAVTTDMSKRTVPTKLEEGEMPLNTYSNKAPFKAKVRSVEKITGPKATGETCHIIIETEGKIPFWEGQSYGVIPPGTKINSKGKEVPTARLYSIASSRYGDDGDGQTASLCVRRAVYVDPETGKEDPAKKGLCSNFLCDATPGTEISMTGPTGKVLLLPADANAPLICVATG
TGIAPFRSFWRRCFIENVPSYKFTGLFWLFMGVGNSDAKLYDEELQAIAKAYPGQFRLDYALSREQNNRKGGKMYIQDKVEEYADEIFDLLDNGAHMYFCGLKGMMPGIQDMLERVAK EKGLNYEEWVEGLKHKNQWHVEVY
gi|1706781|sp|P53991|FENR_CHLRE[1706781]
NCBI: 0gi|1706781|sp|P53991|FENR_CHLRE Ferredoxin NADP
reductase
SIB-BLAST: 0FENR_CHLRE Ferredoxin--NADP reductase, chloroplast
Kazusa: 0U10545.1 Chlamydomonasreinhardtii ferredoxin-NADP+ reductase
Other:
Scores
1
2
Peptides
VNGGPAGEGLDPL-ADDPDTFAELK, bntyl27a_08182004.2232.2236.2.dta, Xcorr 3.64
Gene model: C_1460037 [chlre2:155818]
MAFALATSRKALQVTCKATGKKTAAKARIISYNQIEIAAAPKSSGVEFYGPNRAKWLGPYSENSTPAYLTGEFPGDYGWDTAGLSADPETFKRYRELELIHARWAMLGALGCLTPELLAKSGTKFGEAVWFKAGAQIFSEGGLD
YLGNPSLVHAQNIVATLAVQVILMGLIEGYRVNGGPAGEGLDPLYPGESFDPLGLADDPDTFAELKVKEIKNGRLAMFSMFGFFVQAIVTGKGPVQNLDDHLANPGVNNAFAFATKFTPSA
JGI annotation: Lhcbm4
Scores
NCBI: 2e-136gi|4139216|gb|AAD03731.1|light harvesting complex II
SIB-BLAST: 1e-136tr Q9ZSJ5 CHLRE [Lhcb2]
Kazusa: -
1
Gene model: C_1460037 [chlre2:155818]
MAFALATSRKALQVTCKATGKKTAAKARIISYNQIEIAAAPKSSGVEFYGPNRAKWLGPYSENSTPAYLTGEFPGDYGWDTAGLSADPETFKRYRELELIHARWAMLGALGCLTPELLAKSGTKFGEAVWFKAGAQIFSEGGLD
YLGNPSLVHAQNIVATLAVQVILMGLIEGYRVNGGPAGEGLDPLADDPDTFAELKVKEIKNGRLAMFSMFGFFVQAIVTGKGPVQNLDDHLANPGVNNAFAFATKFTPSA
Altered model is not supported by EST data
Scores
NCBI: 1e-126gi|4139216|gb|AAD03731.1|light harvesting complex II
SIB-BLAST: 1e-126tr Q9ZSJ5 _CHLRE [Lhcb2]
Kazusa: -
2
Example 3 – Alternative splicing?
GenomicPeptideFinder
• Detection of peptides in genomic databases– Increases yield 10 fold when compared to de novo sequencing alone
• Detection of peptides split by introns when deduced from genomic DNA– Enables identification of approximately 20% formerly undetectable
spectra• Gene Annotation
– Enables gene annotation from proteomic data• Additionally
– Detection of proteins by a single supporting peptide significantly enhances identification of
• Short proteins• Low abundant proteins• Modified proteins• Alternatively spliced proteins
– High-Throughput possibility
Allmer et al. (2004) FEBS Lett 562, 202-6.Allmer et al. (2006) Proteomics 6, 6207-6220.
Third generation GPF
• GPF 2: currently in development• improvements:
– also finds intron splits within nucleotide triplets– higher speed
• ~100 times faster than the old GPF• 3 queries per second on average• speedup due to the fact that an index is used and thus
peptides can be “looked up” instead of “searched for”
– includes visualization of hits• could be useful for genome annotation
• a web interface can be found atwww.uni-muenster.de/hippler/gpf
• use gpf as user name and hellofellow as password for the authentication
• GPF 2 is still work in progress, so the service may become temporarily unavailable or move to another location
Third generation GPF
2DB (relational database)
• Replaces PepProtDB– Web based– Multiuser– Fully searchable– Enhanced visualization
www.2db.de.ms
Experiment MS/MS Spectra-Analysis(e.g. SEQUEST)
Import, Filtering,Linkage
DataVisualization
Server
PHP
Local External
Internet
Workflow
Experiment IOverview
Experiment Details
List of all measured Spots / Bands
Spot/Band Labeling
Bild des Experimentsmit Imagemap
Experiment IIOverview
Experiment IIIOverview
MS - Details:• Identified Proteins • Peptide sequences• Link to other DBs• MS²-spectra • Software• Xcorr• ∆Cn• Tools (Plugins)
Spot/Band Details(pI, mass, rT)
Experiment Overview Experiment Details Spot/Band Details
Data export (*.csv)
Imange
ImageMap
Image
Application Structure
Conclusion
• 2DB speeds up data analysis– Automates protein identification– Provides results in Tables– Data organisation facilitates analysis
Acknowledgement
WESTFÄLISCHEWILHELMS-UNIVERSITÄT
MÜNSTER
Frederik Sommer
Christine Markert
Monica Zhang
Joshua Goldenberg
Jens AllmerAndreas BuschMarita Hermann
Susan HawatBernadeta KukuczkaSebastian KuhlgertBianca NaumannElisabeth Ostendorf
Michael SpechtMia Terashima
Michael Hippler
Einar J. Stauber
Blandine Rimbault
FRIEDRICH SCHILLERUNIVERSITÄT
JENA Institute of Plant Biochemistry and Biotechnology
Cooperations
David RoosUPENN, USA
FundingDeutsche ForschungsgemeinschaftResearch Grant UPENN