We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.
Bibliographical noteFunding Information:
This research was supported in part by the National Institutes of Health grants R21AG055377, R01AG052324, R01DK071801, the Clinical and Translational Science Award (CTSA) program, through the NIH National Center for Advancing Translational Sciences (NCATS), grant UL1TR000427, and a Robert Draper Technology Innovation Fund grant with funding provided by the Wisconsin Alumni Research Foundation (WARF). The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531) and Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin−Madison. A.R.B. acknowledges an NIH General Medical Science NRSA Fellowship (1F31GM119365) for funding support. L.L. acknowledges a Vilas Distinguished Achievement Professorship and Janis Apinis Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin−Madison School of Pharmacy.