Derivatization Strategy for Glycomics

Glycomics is the systematic identification and characterization of diverse glycans for their structure, function, activity, and quantity. Global profiling of glycomes of cancer cells, body fluids, or tissues has been considered as an innovative and comprehensive approach to discover and identify biomarkers for various diseases. Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated oligosaccharides were analyzed by matrix-assisted desorption/ionization mass spectrometry (MALDI-MS). In this presentation, I will first discuss a simple and rapid method to modify sialic acids at sialylated oligosaccharides in presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrroli- dinophosphonium hexafluorophosphate (PyAOP). After methylamidation, sialylated oligosaccharides can be analyzed by MALDI-MS without loss of sialic acid moiety. The ESI-MS and MALDI-MS analysis of both 3’- and 6’-sialyllactose derivatives indicated that the quantitatively conversion of sialic acids was achieved, regardless of their linkage types under the optimal derivatization conditions. This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins (Fetuin, human acid glycoprotein and bovine acid glycoprotein) containing different types of complex oligosaccharides. Using this derivatization method, the N-glycans of sera from different species (human, mouse and rat) were successfully characterized by MALDI-MS. Due to the mild reaction conditions, the modification in sialic acid residues can be retained. This improvement makes it possible to detect the sialylated oligosaccharides containing O-acetylated sialic acid moieties using MALDI-MS in positive ion mode.
In the meantime, the development of glycomics is limited by the ability to identify and quantify large numbers of glycans in complex biological samples, which is only partially achieved by current glycomics approaches. I will also present a strategy termed targeted glycomics enabling highly sensitive and consistent identification and quantification of target sets of glycans across multiple samples. The strategy was first applied to the analysis of glycans released from ribonuclease B. A linear dynamic range of three orders of magnitude (0.1-100 ng ribonuclease B) was achieved for the detection of all five glycans. The method exhibited excellent sensitivity, evident by the detection of limit of 100 attomolar for Man9GlcNAc2. The strategy was further demonstrated by characterizing the glycome of pancreas cancer cells. More than 60 N-glycans were identified and detected for about five million cells.