Use of ProteinChip array surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify thymosin beta-4, a differentially secreted protein from lymphoblastoid cell lines

Abstract

The identification of proteins differentially expressed between cancer and normal cells is vital for the development of cancer diagnostics, therapeutics and vaccines. Using a ProteinChip Biomarker System (Ciphergen Biosystems, Fremont, CA) which combines ProteinChip technology with time-of-flight mass spectrometry, we have developed a simple method to screen and identify differentially secreted proteins from tumor cell lines. Mass spectra of the range of proteins secreted from normal B-cells were generated along with those secreted from Epstein-Barr virus transformed B-cells. A mass peak at m/z = 4972.1 that was highly over-represented in the transformed B-cell line was chosen for identification and purified by reversed phase chromatography with concomitant monitoring of fractions by SELDI-TOF MS. The resulting purified protein was digested with trypsin and the peptide masses derived from the SELDI-TOF spectrum were used to search the public databases for protein identification. Fragment matching of the resulting peptides identified the protein as thymosin beta-4. Using LC-electrospray ionization MS/MS, the identity of this protein was confirmed. Thymosin beta-4 is a known marker in LCLs establishing the utility of this method to discover and identify proteins differentially expressed between cancers and their matched normal counterparts.