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

Abstract

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 β-elimination and defined, showing a range of 26 neutral, sulfated, sialylated, and both sulfated and sialylated core 1 (Galβ1-3GalNAcα1-) and core 2 (Galβ1-3­(GlcNAcβ1-6)­GalNAcα1-) 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 Neu5Acα2-3Galβ1-4Glc and Neu5Acα2-6Galβ1-4Glc and for Neu5Acα2-3Galβ1-4GlcNAc and Neu5Acα2-6Galβ1-4GlcNAc linkage isomers with the Neu5Acα2-6 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.

Core-2O-glycans are required for galectin-3 interaction with the osteoarthritis related protein lubricin

“…The PSGL-1/mIgG2b O-linked oligosaccharides generated after transfection of CHO-cells using human core-2 GlcNAcb1-6 glycosyltransferase terminate with sialic acid after adding a LacNAc unit to the natively expressed core 1 structures 40 . This is also the type of core-2 structures found on lubricin 35 .…”

“…O-linked oligosaccharides from lubricin in age-and sex-matched OA patients' and controls' SF were released by reductive β-elimination and analyzed with quantitative MRM on a QTRAP 6500 triple quad-linear ion trap hybrid mass spectrometer as has been described in detail previously 35 . The MRM method was developed to semi-quantify a range of twenty-two core-1 and -2 and -3 glycans on native lubricin from both controls and OA patients 35 . Although the abundances of these glycans were found to differ between the two sample types, the glycan repertoire was the same (Fig.…”

“…The final method included transitions to quantify and identify all 24 identified structures on lubricin. All transition information has been published in detail previously 35 .…”

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking

“…O-linked oligosaccharides from lubricin in age-and sexmatched OA patients' and controls' SF were released by reductive β-elimination and analyzed with quantitative MRM (multiple reaction monitoring) on a QTRAP 6500 triple quadrupolelinear ion trap hybrid mass spectrometer as has been described in detail previously (36). The MRM method was developed to semi-quantify a range of twenty-two core-1 and -2 and -3 glycans on native lubricin from both controls and OA patients (36). We refer to semi-quantification here as, without the appropriate standards, a technical difficulty of O-glycan research, absolute quantification is not attained.…”

“…This is due to differences in ionization and stability between glycans, particularly between charged and neutral glycans. MRM is able to mitigate some of this as each glycan structure, even the sialic acid isomers, are optimized for the conditions that allow for its best quantification (36). Nevertheless, the most apt use of this approach is to compare the same glycan or type of glycans between samples.…”