C HROMOSOME 16 Marta Mendes Ignacio Casal Centro de Investigaciones Biológicas August 2012.

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Presentation transcript:

C HROMOSOME 16 Marta Mendes Ignacio Casal Centro de Investigaciones Biológicas August 2012

P ROTEINS TO SEARCH FOR Cell Lines CCD18 MCF7 Ramos 1D SDS PAGE  g/lane CCD  g/lane RAMOS MCF7 30  g/lane Digestion with trypsin High throughput analysis

LTQ Orbitrap Velos coupled to a Proxeon nanoLC systemE QUIPMENT : LC CONDITIONS Pre column Proxeon ASY-column, L 2cm, ID 100  m, 5  m, C18 ColumnNano separations, NS-AC-11-dp3, L 15 cm, ID 75  m, 3  m, 120 A, Biosphere C18 Mobile phase A0.1%FA/2%ACN Mobile Phase B0.1%FA in ACN Flow rate250 nl/min Gradient Profile160 min from 0 to 35% B MS S YSTEM Low ResolutionHigh Resolution AGC MS13x10 4 1x10 7 AGC MS21x10 4 5x10 4  scan 11 Maximum injection time10500 Isolation width22 Acquisition methodDDA - Top15 Resolution Dynamic exclusion90 s

Q8TBY8-2 CCD18 MCF7 RAMOS H IGH THROUGHPUT DDA T OP 15 No proteins found Proteome discoverer and Sequest with FDR 1% Database: uniprot_sprot_human_HPP PeptideXCorrq-valuePEP KLEEENENLR KLEEENENLR

Q8TBY8 ISOFORMS ALIGNMENT

T ARGET USING GLOBAL MASS LIST LC CONDITIONS Gradient Profile100 min from 0 to 35% B MS S YSTEM Acquisition methodGlobal mass list Cell LineFractionPeptideProteinScorePEP CCD182EFQEEMAALKQ8TBY8; Q8TBY8-2; Q8TBY CCD182LMEGELEALRQ8TBY8; Q8TBY MAXQUANT Proteotypic peptides obtained at SRM Atlas Database: uniprot_sprot_human_HPP

EFQEEMAALK LMEGELEALR

Q8TBY8 isoforms alignment Q8TBY8 isoforms 1 and 2 are present in both CCD18 and Ramos cell lines