Glycoproteomic analysis of human lung adenocarcinomas using glycoarrays and tandem mass spectrometry: differential expression and glycosylation patterns of vimentin and fetuin A isoforms.

ABSTRACT

Human lung cancer is a major cause of cancer mortality worldwide. Advances in pathophysiologic understanding and novel biomarkers for diagnosis and treatment are significant tasks. We have undertaken a comprehensive glycoproteomic analysis of human lung adenocarcinoma tissues. Glycoproteins from paired lung adenocarcinoma and normal tissues were enriched by the lectins Con A, WGA, and AIL. 2-D PAGE revealed 30 differentially expressed protein spots, and 15 proteins were identified by MS/MS, including 8 up- (A1AT, ALDOA, ANXA1, CALR, ENOA, PDIA1, PSB1 and SODM) and 7 down-regulated (ANXA3, CAH2, FETUA, HBB, PRDX2, RAGE and VIME) proteins in lung cancer. By reverse-transcription PCR, nine proteins showed positive correlation between mRNA and glycoprotein expression. Vimentin and fetuin A (alpha(2)-HS-glycoprotein) were selected for further investigation. While for vimentin there was little correlation between total protein and mRNA abundance, expression of WGA-captured glycosylated vimentin protein was frequently decreased in cancer. Glycoarray analysis suggested that vimentins from normal and cancerous lung tissue differ in their contents of sialic acid and terminal GlcNAc. For fetuin A, both total protein and mRNA abundance showed concordant decrease in cancer. WGA- and AIL-binding glycosylated fetuin A was also consistently decreased in cancer. Glycoarray analysis suggested that high mannose glycan structures on fetuin A were only detectable in cancer but not normal tissue. The intriguing expression patterns of different isoforms of glycosylated vimentin and fetuin A in lung cancer illustrate the complexities and benefits of in-depth glycoproteomic analysis. In particular, the discovery of differentially glycosylated protein isoforms in lung adenocarcinoma may represent avenues towards new functional biomarkers for diagnosis, treatment guidance, and response monitoring.