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.
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.
Diamond
Deborah L
DL
Ciphergen Biosystems Inc., Fremont, California, USA.
Zhang
Yanni
Y
Gaiger
Alexander
A
Smithgall
Molly
M
Vedvick
Thomas S
TS
Carter
Darrick
D
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000737:chemistry / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D030562:Databases, Protein; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization; D013947:Thymosin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D000099201:Thymosin beta(4)
D014408:Biomarkers, Tumor; D010446:Peptide Fragments; D013947:Thymosin; D014357:Trypsin; D000099201:Thymosin beta(4)