Glycoproteomic Analysis of Human Lung Adenocarcinomas Using Lectin Glycoarrays and Tandem Mass Spectrometry: Differential Expression and Glycosylation Patterns of Vimentin and Fetuin A Isoforms
MH Roehrl, J-H Rho, JY Wang. Boston Medical Center, Boston, MA; Brigham and Women's Hospital, Boston, MA
Background: Human lung cancer is a major cause of cancer mortality worldwide. Advances in pathophysiologic understanding and novel biomarkers for diagnosis and treatment are sorely needed. We have undertaken a comprehensive glycoproteomic analysis of human lung adenocarcinoma tissues.
Design: The glycoproteomes from paired human lung adenocarcinoma and normal tissue were biochemically enriched by lectin affinity to Con A, WGA, and AIL followed by 2-D PAGE and tandem mass spectrometric (MS/MS) identification. The chemical and structural alterations at N- and O-linked glycosylation sites were interrogated with high-density lectin glycoarrays and analyzed by computational pattern decomposition.
Results: 2-D PAGE analysis revealed 30 differentially expressed protein spots, from which 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 RT-PCR, 9 proteins showed positive correlation between mRNA and glycoprotein expression. Vimentin and fetuin A (α2-HS-glycoprotein) were selected for further investigation. While there was little correlation between total protein abundance and mRNA abundance, expression of Con A-, WGA-, and AIL-captured vimentin protein was consistently decreased in cancer. Lectin glycoarray pattern analysis suggested that vimentin from normal and cancerous lung tissue differ in their contents of sialic acid and GlcNAc. For fetuin A, the correlation between total protein and mRNA abundance showed concordant decrease in cancer. WGA- and AIL-binding fetuin A was also consistently decreased in cancer. Glycoarray analysis suggested that fetuin A glycan structures recognized by Con A were altered in lung cancer, whereas the WGA- and AIL-specific structures were not.
Conclusions: The intriguing expression patterns of different isoforms of glycosylated fetuin A and vimentin in lung cancer illustrate the complexities and benefits of in-depth glycoproteomic analysis. Importantly, mRNA abundance measurements alone fail to capture the chemical diversity of the posttranslationally regulated cancer glycoproteome. The discovery of differentially glycosylated protein isoforms in lung adenocarcinoma may represent avenues towards new functional biomarkers for diagnosis, treatment guidance, and response monitoring.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Tuesday, March 23, 2010 8:30 AM
Platform Session: Section H 1, Tuesday Morning