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 (Kronewitter et al. Anal. Chem. 2014, 86, 6268-6276), 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.

Figure 1

Experimental sample preparation and data analysis pipeline.

Figure 2

Example mass spectra of polysialylated glycans containing 7–11 sialic acids and 1–2 fucose residues. Putative glycan structures are shown and the compositions are consistent with number of sialic acid groups exceeding the possible number of N-glycan antennae. The glycan structure depictions are constant with the CFG nomenclature where the squares = N-acetylhexosamine, circles = hexose, triangles = fucose, and diamonds = sialic acid.

Figure 3

GlycoGrid Comparison between the current glycan library that allowed for polysialic acid and lactonization (orange) and the former library that did not (black). The regions circled indicated several polysialylated glycans that differed by either a sialic acid or a fucose residue. Blue horizontal lines indicate regions where polysialylated glycans were plotted.

Figure 4

High-resolution HCD spectrum of fragments from N-glycan containing polysialic acid. Inset: high-resolution MS1 spectrum indicating monoisotopic mass and a putative cartoon of the glycan structure. CFG nomenclature was used to denote mass differences with the following modification: Open circle symbols represent generic hexose mass differences.

Figure 5

In-source fragmentation detected linking Hex₄HexNAc₇Neu5Ac₃ (Lactone₂) and Hex₄HexNAc₇Neu5Ac₄ (Lactone₂) by one Neu5Ac residue. The first chromatographic peak shown on the left between the vertical lines had a correlation coefficient of 0.999 after the peaks were modeled and correlated. The inlayed mass spectrum corresponded to the high-resolution isotope profiles of the parent (orange) and fragment (gray) glycans. Cartoon structures (CFG nomenclature) of the parent and fragment illustrate the in-source fragmentation loss of a sialic acid.

Figure 6

Percent lactonization (number of lactone bonds divided by the number of sialic acid residues) distribution of all “heavily” sialylated glycans. Orange denotes glycans with no lactonization detected. Light gray indicates glycans with 1–2 lactones. Dark gray indicates glycans with more than 2 lactones.

Figure 7

Extending MS/MS fragmentation data with glycan families and accurate mass.

Figure 8

Extracted ion chromatograms (m/z 1040.89) showed the quantitative reduction to alditol form achieved by quantitative hydrolysis of β-glycosylamine groups first. 99.7% conversion to the alditol form was achieved.

Figure 9

Number of lactones detected per “heavily” sialylated glycan.

Figure 10

Number of sialic acid residues per glycan. Orange denotes “heavily” sialylated glycans. Gray includes typical monosialylation on the terminus of the galactose residue. Nonsialylated glycans, such as high mannose glycans, are included in the 0 column.