Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations, and Electrospray Ionization-Mass Spectrometry

Abstract

The N-glycan diversity of human serum glycoproteins, i.e., the human blood serum N-glycome, is both complex and constrained by the range of glycan structures potentially synthesizable by human glycosylation enzymes. The known glycome, however, has been further limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to include polysialylated N-glycans. Sample preparation improvements included acidified, microwave-accelerated, PNGase F N-glycan release to promote lactonization, and sodium borohydride reduction, that were both optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. Online separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient, providing additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) was utilized. When these improved methods are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described (24881670Anal. Chem.20148662686276), we are able to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrated the application of these advances in the context of the human serum glycome, and for which our initial observations included the detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…Kronewitter et al () have released glycans from serum glycoproteins using PNGase F acidified to pH 5.5 with HCl in a microwave reactor, under which conditions lactonization of the sialic acids occurred. Combined with other modifications to the standard clean‐up methods, the authors were able to report the identification of a range of highly sialylated glycans including those with polysialic acid.…”

Mass spectrometry for protein sialoglycosylation

“…A modified ESI, termed subambient pressure ionization with nanoelectrospray (SPIN), has been applied for profiling (poly)sialylated N ‐glycans from human serum sample . SPIN source has a series of advantages when compared to conventional ESI.…”

A Method for In-Depth Structural Annotation of Human Serum Glycans That Yields Biological Variations

“…Even highly sialylated species are eluted as shown recently by Kronewitter et al with glycans containing as many as 11 sialic acids. 34 …”