Multiplexed Surrogate Analysis of Glycotransferase Activity in Whole Biospecimens

Dysregulated glycotransferase enzymes in cancer cells
produce aberrant
glycanssome of which can help facilitate metastases. Within
a cell, individual glycotransferases promiscuously help to 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 gas chromatography–mass
spectrometry (GC-MS) run. Numerous nonliver organ cancers are known
to induce the production of abnormally glycosylated serum proteins.
Thus, following analytical validation, in blood plasma, the technique
was applied to a group 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 (CEE) study to gauge the
clinical utility of the approach toward the detection of lung cancer.
Ten smoking-independent glycan node ratios were found that detect
lung cancer with individual receiver operating characteristic (ROC)
c-statistics ranging from 0.76 to 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.