FIG. 3.

SELDI-TOF MS identification of 28.9-kDa candidate biomarker. (A) The 28.9-kDa protein was enriched from serum samples by anion-exchange chromatography (flowthrough) and protein A chromatography (flowthrough), concentrated/desalted using YM30 filtration, and finally purified by 12% Bis-Tris SDS-PAGE. Coomassie blue-stained bands were extracted and reprofiled on NP20 arrays. A band corresponding to the 28.9-kDa protein was digested in solution with trypsin, and the digest was analyzed by tandem mass spectrometry. Seven peptides were identified as components of the N-terminal fragment of fibronectin. (B) Sequence of the first 300 amino acids of human fibronectin. The full-length sequence of fibronectin, consisting of 2,355 amino acids, is not shown. The fragments identified by MS/MS are highlighted in bold. Importantly, one fragment with m/z 1,707.78 Da had a nontryptic cut at the C terminus, indicating that this was the C-terminal end of the 28.9-kDa protein. The position of the peptide used to generate the rabbit antiserum is underlined. The calculated molecular mass of the sequence from the N terminus to Val258, with the N-terminal Gln modified to pyrrolidone carboxylic acid and with nine disulfide bonds, is 28,931 Da. The putative C-terminal Val258 is bold and underlined.