Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.
Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
B E
BE
Chong
D M
DM
Lubman
A
A
Rosenspire
F
F
Miller
D002461:Cell Line, Transformed / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051518:Fibroblast Growth Factor 3; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000302:isolation & purification; D005348:Fibrocystic Breast Disease / Q000473:pathology; D006801:Humans; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000302:isolation & purification; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000302:isolation & purification; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000302:isolation & purification
D051518:Fibroblast Growth Factor 3; D005346:Fibroblast Growth Factors; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D016283:Proto-Oncogene Proteins p21(ras); D018719:Erb-b2 Receptor Tyrosine Kinases; D011988:Receptors, Thyroid Hormone; D016159:Tumor Suppressor Protein p53; C496444:FGF3 protein, human; C501009:HRAS protein, human