Two-dimensional gel database of human breast carcinoma cell expressed proteins: an update.
Previously, we reported a two-dimensional gel map and database with molecular weight/isoelectric point (Mr/pI) loci for 22 proteins expressed in the breast carcinoma cell line, MDA-MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588-598). Here we update this database with Mr/pI loci for a further nine cytoplasmic proteins and three Triton X-114 solubilised membrane proteins from MDA-MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X-114 soluble protein and assigned an Mr/pI locus. During the course of isolating proteins from the Triton X-114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier ampholytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS-polyacrylamide gels after the use of carrier ampholytes for the first dimension. We also report our mass spectrometric-based procedure for identifying two-dimensional electrophoresis (2-DE) gel-resolved proteins, combining in-gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping and electrospray ionisation--ion trap--mass spectrometry.
Rasmussen
R K
RK
Trescowthick Research Center, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Ji
H
H
Eddes
J S
JS
Zugaro
L M
LM
Reid
G E
GE
Simpson
R J
RJ
Dorow
D S
DS
D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000737:chemistry*; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis*; D017830:Octoxynol; D011092:Polyethylene Glycols; D012995:Solubility; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40