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 spectrometr y (MALDI-TOFMS) has been used to rapidly profile the protein content of hum an 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 br east epithelium to fully malignant cells, Distinct differences in the prote in profiles were observed with progression, and specific proteins associate d 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 u sing non-porous reversed-phase high performance liquid chromatography (NP-R P-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight inform ation without interference from other proteins in the whole cell lysates, a nd to avoid suppression effects in mixtures of proteins detected by MALDI-T OFMS. In order to confirm the identity of these oncoproteins, the cell lysa tes were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for furt her analysis, Trypsin and cyanogen bromide digestions were performed on the se proteins eluted from excised PVDF bands which were then analyzed by MALD I-TOFMS. The identity of these proteins was confirmed by database matching procedures, (C) 1998 John Wiley & Sons, Ltd.