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

• Published in: Rapid communications in mass spectrometry Vol. 12; no. 24; pp. 1986 - 1993
• Main Authors: Chong, Bathsheba E., Lubman, David M., Rosenspire, Allen, Miller, Fred
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 30.12.1998
• Subjects: Boulder; Cell Line, Transformed - chemistry; Chestnut Ridge; Chromatography, High Pressure Liquid; COMPANY Beckman; COMPANY Beckman, Fullerton, CA,USA; COMPANY Continuum; COMPANY Continuum, Santa Clara, CA, USA; COMPANY LeCroy Corp; COMPANY LeCroy Corp., Chestnut Ridge, NY, USA; COMPANY Micra; COMPANY Micra, Northbrook, IL, USA; COMPANY R.M. Jordan Co; COMPANY R.M. Jordan Co., Grass Valley, CA, USA; COMPANY Timberline; COMPANY Timberline, Boulder, CO, USA; Electrophoresis, Polyacrylamide Gel; Female; Fibroblast Growth Factor 3; Fibroblast Growth Factors - chemistry; Fibroblast Growth Factors - isolation & purification; Fibrocystic Breast Disease - pathology; Fullerton; Grass Valley; Humans; Molecular Weight; Neoplasm Proteins - chemistry; Neoplasm Proteins - isolation & purification; Northbrook; PRODUCT HPLC Column; PRODUCT LeCroy 9310 Am digital Oscilloscope; PRODUCT Minilite 10 Hz Nd:YAG laser System; PRODUCT System Gold® HPLC; PRODUCT Timberline column heater; PRODUCT TOF Mass Spectrometer; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - isolation & purification; Proto-Oncogene Proteins c-myc - chemistry; Proto-Oncogene Proteins c-myc - isolation & purification; Proto-Oncogene Proteins p21(ras) - chemistry; Proto-Oncogene Proteins p21(ras) - isolation & purification; Receptor, ErbB-2 - chemistry; Receptor, ErbB-2 - isolation & purification; Receptors, Thyroid Hormone - chemistry; Receptors, Thyroid Hormone - isolation & purification; Santa Clara; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - instrumentation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Tumor Cells, Cultured - chemistry; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - isolation & purification; USA
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/(SICI)1097-0231(19981230)12:24<1986::AID-RCM419>3.0.CO;2-H

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. Copyright © 1998 John Wiley & Sons, Ltd.