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 >or=.9 and >or=.5, respectively. The specificity for the N-glycomotif was 88% at P >or= .9. Penetration into the moderate to low protein concentration range (microg-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 mug to ng/mL range. Pleural effusion is a useful source for biomarker research in lung cancer.
Soltermann
Alex
A
Institute for Surgical Pathology, University Hospital Zurich, Zurich Switzerland. alex.soltermann@usz.ch
Ossola
Reto
R
Kilgus-Hawelski
Sandra
S
von Eckardstein
Arnold
A
Suter
Tobias
T
Aebersold
Ruedi
R
Moch
Holger
H
D000230:Adenocarcinoma / Q000737:chemistry*; D000368:Aged; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis*; D002853:Chromatography, Liquid; D005260:Female; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000737:chemistry*; D008297:Male; D010996:Pleural Effusion / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry; D014407:Tumor Cells, Cultured
D015295:Antigens, Tumor-Associated, Carbohydrate; D006023:Glycoproteins; D020543:Proteome