FIRST APPROACH TO THE SHOTGUN UNKNOWN PROTEINS

sHPP CHROMOSOME 16 MEETING

August, 28, 2012La 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

250150

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.