Glycan Node Analysis: A Bottom-up Approach to Glycomics

Abstract

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides (“glycan nodes”) present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

A spin column-free approach to sodium hydroxide-based glycan permethylation

“…One spin column permethylation procedure with 10 min reaction time (SC 10 min) was developed by Kang et al , 7 Goetz et al 24 and further modified by Borges et al , 19 the details of which can be found in their recently published work. A second spin column permethylation procedure with 50 min reaction time (SC 50 min) was summarized in recent work of Hu et al 20 2-Acetamido-2-deoxy-D-lactose (Gal-4GlcNc, a.k.a.…”

“…19 Sodium hydroxide beads were collected in a Pierce spin column (900 μL volume) up to 5 mm below the rim, and washed once with 350 μL of acetonitrile (ACN) followed by two rinses with 350 μL of DMSO. Nine microliters (9 μL) of aqueous glycan standard solution or a whole biofluid sample ( e.g.…”

“…18,19 Two molar TFA was prepared and added (325 μL) to each test tube and allowed to react for 2 h at 120 °C. Samples were dried under a gentle stream of nitrogen at 73 °C.…”

“…For routine relative quantification of each glycan “node” from glycan standards or blood plasma, the 2–4 most abundant and/or diagnostic fragment ions 18,19 were summed using a 0.15 Da extracted ion chromatogram mass window for quantification. The summed extracted ion chromatogram (XIC) peak areas were automatically integrated and quantified, then exported to a spreadsheet for further calculation.…”

“…1). 18,19 With the additional steps, O-linked glycans, N-linked glycans, and glycolipids are all released and ultimately converted into partially methylated alditol acetates (PMAAs, Fig. 2) which are highly amenable to analysis by gas chromatography-mass spectrometry (GC-MS).…”