Abstract 5568: Development of serum glycoproteomic profiling technology for the identification of pancreatic cancer biomarkers: simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes

Abstract

Abstract Glycosylation is one of the most important and abundant post-translational modifications in nature. Glycoproteins play important roles during molecular and cellular recognition in development, growth, and cellular communication and in particular are involved in cancer progression. Glycoproteins have been used as therapeutic targets and biomarkers for cancer prognosis, diagnosis, and monitoring. As mass spectrometry-based systems biology begins to revolutionize our understanding of biomedical sciences, the ability to efficiently and comprehensively profile glycoproteins in biological samples of interest (body fluids, cell surface, etc.) is critical to many biological and clinical researchers. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin column chromatography (IGEL). It is based on glycan structure-specific enrichment of glycopeptides by lectin-column chromatography and site-directed tagging of N-glycosylation sites by 18O during the elution with N-glycosidase. The N-glycosidase elution in the presence of H218O results in the specific amino acid substitution, asparagine to 18O-asparatic acid residue, with 3-Da increase of the peptide molecular weight. The mass spectrometric identification of this 3-Da increase enables absolutely accurate determination of N-glycosylation sites in complicated samples like serum. Furthermore, the combination of IGEL with label-free quantification system Expressionist enabled us not only to identify N-glycosylation sites accurately but also to quantitatively compare glycan structures on each glycosylation site in a single LC/MS/MS analysis. We applied the IGEL method to the comparative analysis of 18 serum samples from 4 healthy controls, 4 chronic pancreatitis patients, 5 stage-I pancreatic cancer patients, and 5 stage-IV pancreatic cancer patients on the Sambucus sieboldiana agglutinin (SSA) lectin column recognizing alpha 2, 6-linked sialic acid residues. QSTAR-Elite tandem mass spectrometry analysis of SSA-IGEL purified serum samples determined 121 individual N-glycosylation sites captured by the SSA lectin. The further t-test and principal component analysis with Expressionist label-free quantification system revealed that the stage-I pancreatic cancer patients group was statistically distinguishable from normal and pancreatitis groups by the affinity changes of 12 glycosylation sites to SSA lectin. This fact indicated that these glycosylation sites should be potential carbohydrate-targeting biomarkers for pancreatic cancer. Thus our quantitative glycoproteomic technology could be extremely valuable for the identification of carbohydrate biomarkers from various body fluid specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5568.