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This study used a surface enhanced desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) discovery platform to generate protein expression profiles in search of overexpressed proteins in lung tumors as potentially novel molecular targets. Two differentially expressed protein peaks at m/z 4219 and 7762 in the SELDI-TOF spectra were identified in lung tumor specimens as macrophage migration inhibitory factor .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 trypsin. Using QSTAR MS/MS, the identity of this protein was confirmed. The study were performed using serum samples or tissue specimens from 146 patients with lung adenocarcinoma (including smoker and nonsmoker), 72 patients with lung squamous cell carcinoma, 40 patients with resectable non-small cell lung cancer (NSCLC), and 300 control volunteers serum, 23 patients with benign lung diseases. The group of healthy controls consisted of 100 age-and sex-matched individuals without known malignant disease. The most statistically significant differentially expressed peaks in the tumor as compared with the normal serum were found at m/z 4,219 and 7762 (±0.1% mass accuracy). Using a classification scheme based on our mass spectral analyses of the original set of 103 tumor/normal serum pairs, we classified the blinded serum from these additional 30 patients. Overall, both of the distinguishing ion signals were seen in 27 of 34 serum and in only 1 of 40 nonmalignant serum (84.3% sensitivity, 87.4% specificity). Ion signals at either m/z 4,219 or 7,762 were found in 30 of 34 serum and 3 of 40 nonmalignant serum (88% sensitivity, 93% specificity). When examined individually, the peak at m/z 12,338 was seen in 30 of 34 serum and 3 of 40 nonmalignant (88% sensitivity, 93% specificity). The peak at m/z 17,882 was found in 27 of 34 tumor and 1 of 40 nonmalignant (79% sensitivity, 98% specificity).A mass spectrometry-based peptide mapping strategy was used to identify the proteins producing the ion signals at m/z 4,219 and 7,762. Ultimately, several proteins in spots that had migration distances consistent with 4,219 and 7,762 kDa proteins were subjected to an in-gel tryptic digestion. The resulting peptides from each protein digest were sequenced by mass spectrometry. The identification was substantiated by MS/MS data on the following 8 tryptic peptides: LAVLLDNILGRIGKLESKVDNLVVNGTGTNSTNST,etal; .These peptides spanned 40% of CyP-A’s linear amino acid sequence. MIF was identified based on MS/MS data from one tryptic peptide and on mapping the mass of an orher tryptic peptide to the following linear sequence in MIF: LCNALLYGGAYPPPCKKELAASLALGLELSER; etal.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]