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FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS
sHPP CHROMOSOME 16 MEETING
August, 28, 2012
La Cristalera, Miraflores de la Sierra
INDEX• ASSIGNED PROTEINS
• MATERIALS AND METHODS
• RESULTS:– MCF7 OFF GEL FRACTIONS– RAMOS CELL LINE ENTIRE EXTRACT RESULTS
• CONCLUSIONS AND FUTURE PERSPECTIVES
KNOWN PROTEINS ASSIGNED TO THE UCM-PCM PROTEOMICS UNIT
ENSEMBLE ID ACCESSION NUMBER GENE ID PROTEIN ID
ENSG00000184471 P60827 C1QT8_HUMAN C1QTNF8
ENSG00000005513 P57073 SOX8_HUMAN SOX8
ENSG00000048462 Q02223 TNR17_HUMAN TNFRSF17
ENSG00000140798 Q96J65 MRP9_HUMAN ABCC12
ENSG00000166743 Q08AH1 ACSM1_HUMAN ACSM1
ENSG00000140948 Q8WYQ9 ZCH14_HUMAN ZCCHC14
MATERIALS AND METHODS
SEVERAL TYPES OF SAMPLES
• 3 CELL LINES: – Ramos cell line (Ph. D. Fuentes)– MCF7 cell line (Ph. D. Canals)– CCD18 cell line (Ph. D. Casals)
• OFF-GEL SAMPLES: – ONLY FOR MCF-7 CELL LINE.– 12 fractions, following the provided protocol.
• IN SOLUTION DIGESTIONS OF THE PROTEIN EXTRACT: – Usual own lab protocol. – Digestion for the entire extracts 3 lines. Only Ramos cells were
used to search our unknown proteins.
PROTEINS AND PEPTIDES SEARCH
• PROTEIN SEQUENCES USING SWISSPROT ACCESSION NUMBER. (http://www.uniprot.org/)
• ENSEMBL ENTRANCES: (http://www.ensembl.org/index.html)
• NCBI BLAST TO SEARCH PROTEOTYPIC PEPTIDES. (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins)
• MATCHING OF PEPTIDES FOR ACQUISITION METHOD:– GPM AND NIST SPECTRA DATABASES FOR SKYLINE SOFTWARE.
NO RESULTS WERE FOUND.
– SKYLINE DIGESTION AND UNIQUE PEPTIDES SEARCH USING THE BACKGROUND PROTEOME.
– MRM PILOT.
EQUIPMENTS AND SOFTWARES
• PREDICTOR SOFTWARES:– SRM ATLAS (http://www.srmatlas.org/)– ESP PREDICTOR: not good predictions
• EQUIPMENT: – AB SCIEX QTRAP 5500 (CNB)– QTRAP RUNNING CONDITIONS: 60 min. gradient.
• Protocol: 1 protein per running in the analyst method.
– Agilent Technologies 3100 Offgel Fractionator.• Protocol: WE FOLLOWED THE PROVIDED PROTOCOL.
• PROCESSING SOFTWARES: – SKYLINE (v. 1.2.0.3425 MacCoss Lab Software)
(https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view)– MRM PILOT (AB SCIEX 2.1.673.0 version)– ANALYST (AB SCIEX 1.6.0 version)
PROTEIN MRM PILOT/ANALYST ACQUISITION PARAMETERS
PROTEIN ID TRANSITIONS PEPTIDES DWELL TIME OBSERVATIONS
C1QTNF8 70 5 15 7-19 res. 1 miss, M, variable mod.
SOX8 72 7 20 8-15 res. 1 miss
TNFRSF17 59 4 20 7-19 res. 1 miss, M, variable mod.
ABCC12 95 12 10 8-15 res. 0 miss
ACSM1 98 11 10 7-19 res. 0 miss
ZCCHC14 112 11 10 8-15 res. 0 miss
MCF7 CELLS RESULTS
OFF-GEL FRACTIONS
MCF-7 10% ACRYLAMIDE SDS PAGE SAMPLE
MW MW IEF FRACTIONSMCF7 SAMPLE
250
150
100
75
50
10
15
25
37
RAMOS CELL LINE RESULTS
MRM DISCOVERY
POSSIBLE (but not confirmed) PEPTIDES IN SAMPLES
PROTEIN ID Nº ANALYST METHOD PEPTIDES
3 OR MORE TRANS. PEPTIDES 2 TRANS. PEPTIDES
C1QTNF8 5 1 0
SOX8 7 1 0
TNFRSF17 4 1 1
ABCC12 10 0 0
ACSM1 11 0 3
ZCCHC14 11 1 0
CONCLUSIONS AND FUTURE PERSPECTIVES
• OFF GEL: BAD PROTEIN SEPARATION.
• QTRAP DIDN´T WORK FORTHE ENTIRE RAMOS EXTRACT.
• PREVIOUS SEPARATION OF THE SAMPLE .
• CARRY OUT PREVIOUS SHOTGUN WITH OFF GEL FRACTIONS.
• CHECK QTRAP WITH THE OFFGEL MCF7 FRACTIONS.
• OPTIMIZE PARAMETERS AND TO SOLVE PROBLEMS.
• SYNTHETIC PEPTIDES.
• NORMALIZATION OF THE METHODS.