N-glycoprotein profiling of lung adenocarcinoma pleural effusions by shotgun proteomics

BACKGROUND. Malignant pleural effusion of advanced lung adenocarcinoma may be a valid source for detection of biomarkers, such as N-glycosylated proteins (N-GP), because tumor cells grow during weeks in this liquid. The authors aimed for creation of N-GP effusion profiles from routine cytology specimens to detect relevant biomarkers. METHODS. Hundred microliters of malignant pleural effusions of 5 patients with lung adenocarcinoma and 5 nonmalignant controls were used for triplicate N-GP capture by solid-phase extraction. After trypsin digest and PNGase F release, a liquid chromatography separation connected online to a tandem mass spectrometer was performed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS. In the total of 10 samples, 170 and 278 nonredundant proteins were detected with probabilities of >=.9 and >=.5, respectively. The specificity for the N-glycomotif was 88% at P >=.9. Penetration into the moderate to low protein concentration range (mu g-ng/mL) occurred, and several proteins associated with tumor progression or metastasis were identified, including CA-125, CD44, CD166, lysosome-associated membrane glycoprotein 2 (LAMP-2), multimerin 2, and periostin. MS identifications were correlated with the corresponding immunoreactivity in either effusion fluid or tumor tissue. CONCLUSIONS. in conclusion, reduction of sample complexity by N-GP capturing allows detection of proteins in the mu g to ng/mL range. Pleural effusion is a useful source for biomarker research in lung cancer.