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Deciphering Isomers with a Multiple Reaction Monitoring Method for the Complete Detectable O-Glycan Repertoire of the Candidate Therapeutic, Lubricin

Journal article
Authors Sarah A. Flowers
C. S. Lane
Niclas G. Karlsson
Published in Analytical Chemistry
Volume 91
Issue 15
Pages 9819-9827
ISSN 0003-2700
Publication year 2019
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 9819-9827
Language en
Keywords sea-urchin sperm, absolute quantitation, recombinant lubricin, salivary, muc7, unicarb-db, in-vitro, protein, glycosylation, acid, identification, Chemistry
Subject categories Biochemistry and Molecular Biology, Medical Biotechnology


Glycosylation is a fundamental post-translational modification, occurring on half of all proteins. Despite its significance, our understanding is limited, in part due to the inherent difficulty in studying these branched, multi-isomer structures. Accessible, detailed, and quantifiable methods for studying glycans, particularly O-glycans, are needed. Here we take a multiple reaction monitoring (MRM) approach to differentiate and relatively quantify all detectable glycans, including isomers, on the heavily O-glycosylated protein lubricin. Lubricin (proteoglycan 4) is essential for lubrication of the joint and eye. Given the therapeutic potential of lubricin, it is essential to understand its O-glycan repertoire in biological and recombinantly produced samples. O-Glycans were released by reductive beta-elimination and defined, showing a range of 26 neutral, sulfated, sialylated, and both sulfated and sialylated core 1 (Gal beta 1-3GalNAc alpha 1-) and core 2 (Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha 1-) structures. Isomer-specific MRM transitions allowed effective differentiation of neutral glycan isomers as well as sulfated isomeric structures, where the sulfate was retained on the fragment ions. This strategy was not as effective with labile sialylated structures; instead, it was observed that the optimal collision energy for the m/z 290.1 sialic acid B-fragment differed consistently between sialic acid isomers, allowing differentiation between isomers when fragmentation spectra were insufficient. This approach was also effective for purchased Neu5Ac alpha 2-3Gal beta 1-4Glc and Neu5Ac alpha 2-6Gal beta 1-4Glc and for Neu5Ac alpha 2-3Gal beta 1-4GlcNAc and Neu5Ac alpha 2-6Gal beta 1-4GlcNAc linkage isomers with the Neu5Ac alpha 2-6 consistently requiring more energy for optimal generation of the m/z 290.1 fragment. Overall, this method provides an effective and easily accessible approach for the quantification and annotation of complex released O-glycan samples.

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Utskriftsdatum: 2020-08-14