Glycoproteomic Analysis of Bronchoalveolar Lavage (BAL) Fluid Identifies Tumor-Associated Glycoproteins from Lung Adenocarcinoma

Cytological
examination of cells from bronchoalveolar lavage (BAL)
is commonly used for the diagnosis of lung cancer. Proteins released
from lung cancer cells into BAL may serve as biomarkers for cancer
detection. In this study, N-glycoproteins in eight
cases of BAL fluid, as well as eight lung adenocarcinoma tissues and
eight tumor-matched normal lung tissues, were analyzed using the solid-phase
extraction of N-glycoprotein (SPEG), iTRAQ labeling,
and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of
80 glycoproteins found in BAL specimens, 32 were identified in both
cancer BAL and cancer tissues, with levels of 25 glycoproteins showing
at least a 2-fold difference between cancer and benign BAL. Among
them, eight glycoproteins showed greater than 2-fold elevations in
cancer BAL, including Neutrophil elastase (NE), Integrin alpha-M,
Cullin-4B, Napsin A, lysosome-associated membrane protein 2 (LAMP2),
Cathepsin D, BPI fold-containing family B member 2, and Neutrophil
gelatinase-associated lipocalin. The levels of Napsin A in cancer
BAL were further verified in independently collected 39 BAL specimens
using an ELISA assay. Our study demonstrates that potential protein
biomarkers in BAL fluid can be detected and quantified.

支气管肺泡灌洗（bronchoalveolar lavage, BAL）所得细胞的细胞学检查，是肺癌诊断的常用手段。肺癌细胞释放入BAL的蛋白质，可作为癌症检测的生物标志物。本研究采用固相萃取N-糖蛋白法（solid-phase extraction of N-glycoprotein, SPEG）、iTRAQ标记法及液相色谱-串联质谱（liquid chromatography tandem mass spectrometry, LC-MS/MS），对8例BAL液标本、8例肺腺癌组织标本及8例肿瘤配对正常肺组织标本中的N-糖蛋白（N-glycoprotein）进行了分析。在BAL标本检出的80种糖蛋白中，32种同时在癌性BAL与癌组织中被鉴定；其中25种糖蛋白的表达水平在癌性与良性BAL间至少存在2倍差异。在上述差异糖蛋白中，8种在癌性BAL中呈现超过2倍的上调，包括中性粒细胞弹性蛋白酶（Neutrophil elastase, NE）、整合素α-M（Integrin alpha-M）、Cullin-4B、天冬氨酸蛋白酶A（Napsin A）、溶酶体相关膜蛋白2（lysosome-associated membrane protein 2, LAMP2）、组织蛋白酶D（Cathepsin D）、BPI折叠家族B成员2（BPI fold-containing family B member 2）及中性粒细胞明胶酶相关脂质运载蛋白（Neutrophil gelatinase-associated lipocalin）。本研究进一步采用酶联免疫吸附试验（ELISA），对独立收集的39例BAL标本中的Napsin A水平进行了验证。本研究证实，BAL液中存在可被检测与定量的潜在蛋白质生物标志物。