Multiplexed Surrogate Analysis of Glycotransferase Activity in Whole Biospecimens

Abstract

Dysregulated glycotransferase enzymes in cancer cells produce aberrant glycans—some of which can help facilitate metastases. Within a cell, individual glycotransferases promiscuously help construct dozens of unique glycan structures, making it difficult to comprehensively track their activity in biospecimens—especially where they are absent or inactive. Here we describe an approach to deconstruct glycans in whole biospecimens then analytically pool together resulting monosaccharide-and-linkage-specific degradation products (“glycan nodes”) that directly represent the activities of specific glycotransferases. To implement this concept a reproducible, relative quantitation-based glycan methylation analysis methodology was developed that simultaneously captures information from N−, O−, and lipid linked glycans and is compatible with whole biofluids and homogenized tissues; in total over 30 different glycan nodes are detectable per GC-MS run. Numerous non-liver organ cancers are known to induce production of abnormally glycosylated serum proteins. Thus following analytical validation in blood plasma the technique was applied to a cohort of 59 lung cancer patient plasma samples and age/gender/smoking-status-matched non-neoplastic controls from the Lung Cancer in Central and Eastern Europe Study to gauge the clinical utility of the approach towards detection of lung cancer. Ten smoking-independent glycan node ratios were found that detect lung cancer with individual ROC c-statistics ranging from 0.76–0.88. Two glycan nodes provided novel evidence for altered ST6Gal-I and GnT-IV glycotransferase activities in lung cancer patients. In summary, a conceptually novel approach to the analysis of glycans in unfractionated human biospecimens has been developed that, upon clinical validation for specific applications, may provide diagnostic and/or predictive information in glycan-altering diseases.

Glycan Node Analysis: A Bottom-up Approach to Glycomics

“…, 2,4-Man, 2,6-Man, 3,4,6-Man, and 3,4-GlcNAc, Figure 2). 33 Given the manner in which XIC peak areas of glycan nodes are normalized (Step 6.3), as long as the relative yield of each individual HexNAc remains consistent relative to other HexNAcs (which it does, see Table 1), useful information on the relative quantities of the different HexNAcs between different samples can be obtained. This holds true for hexoses as well.…”

“…Target ions for each PMAA have been published elsewhere, 33 but the following alterations have been made: t-Glc ions are now 145.1 + 205.1; 2-Man ions are 161.1 + 189.1; 3-Gal ions are 161.1 + 233.1, 6-Gal ions are 161.1 + 189.1 + 233.1; 2,6-Man ion is 189.1; 3,6-Man ions are 189.1 + 233.1.…”

“…Because each glycosyltransferase may operate on more than one glycoconjugate substrate and across different growing glycan polymers, deregulated biosynthetic cascades yield disproportionally increased amounts of only one glycan product but several heterogeneous classes of aberrant glycans in intra- or extracellular fluids. 33 However, such unique aberrant glycans are sometimes considered impractical as biomarkers for cancer or other glycan-affective pathologies because, compared to the large pool of well-regulated glycans, these aberrant glycans represent a very small fraction that may often remain undetectable even by such highly sensitive techniques as mass spectrometry. For example, in intra- and extracellular body fluids, the broad protein-concentration spectrum (which spans eight orders of magnitude) can prevent detection of scarce glycoproteins that are masked by the more abundant species.…”

“…We modified this technique in 2013 and adapted it for whole unfractionated biofluids and homogenized tissue samples by incorporating trifluoroacetic acid (TFA) hydrolysis, reduction, and acetylation steps. 33 These additional steps also release glycans from glycolipids and N -linked glycans from glycoproteins and convert them into partially methylated alditol acetates (PMAAs, Figure 1), whose distinctive methylation-and-acetylation patterns facilitate analysis by GC-MS and uniquely characterize the constituent glycan nodes in the original intact glycan polymer 41 (Figure 2). 33 Ultimately, this procedure produces a composite portrait of all the glycans in a complex biofluid based on direct, relative quantification of unique glycan features such as “core fucosylation”, “6-sialylation”, “bisecting GlcNAc”, and “beta 1–6 branching”-each derived from a single GC-MS chromatographic peak.…”

“…33 These additional steps also release glycans from glycolipids and N -linked glycans from glycoproteins and convert them into partially methylated alditol acetates (PMAAs, Figure 1), whose distinctive methylation-and-acetylation patterns facilitate analysis by GC-MS and uniquely characterize the constituent glycan nodes in the original intact glycan polymer 41 (Figure 2). 33 Ultimately, this procedure produces a composite portrait of all the glycans in a complex biofluid based on direct, relative quantification of unique glycan features such as “core fucosylation”, “6-sialylation”, “bisecting GlcNAc”, and “beta 1–6 branching”-each derived from a single GC-MS chromatographic peak. This article presents further optimization of the permethylation, acetylation, isolation, and clean-up stages along with improvements in the mode of relative quantification.…”