Journal
MOLECULAR & CELLULAR PROTEOMICS
Volume 18, Issue 1, Pages 28-40Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.RA118.000906
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Funding
- National Cancer Institute (Early Detection Research Network) [U01CA152653]
- National Institute of General Medical Sciences [1R41GM112750]
- National Institute of Allergy and Infectious Disease [R21AI129872]
- National Institutes of Health [P41GM10349010, 1S10OD018530]
- NATIONAL CANCER INSTITUTE [U01CA152653, U01CA168896] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R21AI129872] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R42GM112750] Funding Source: NIH RePORTER
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The difficulty in uncovering detailed information about protein glycosylation stems from the complexity of glycans and the large amount of material needed for the experiments. Here we report a method that gives information on the isomeric variants of glycans in a format compatible with analyzing low-abundance proteins. Onchip glycan modification and probing (on-chip gmap) uses sequential and parallel rounds of exoglycosidase cleavage and lectin profiling of microspots of proteins, together with algorithms that incorporate glycan-array analyses and information from mass spectrometry, when available, to computationally interpret the data. In tests on control proteins with simple or complex glycosylation, on-chip gmap accurately characterized the relative proportions of core types and terminal features of glycans. Subterminal features (monosaccharides and linkages under a terminal monosaccharide) were accurately probed using a rationally designed sequence of lectin and exoglycosidase incubations. The integration of mass information further improved accuracy in each case. An alternative use of on-chip gmap was to complement the mass spectrometry analysis of detached glycans by specifying the isomers that comprise the glycans identified by mass spectrometry. On-chip gmap provides the potential for detailed studies of glycosylation in a format compatible with clinical specimens or other low-abundance sources.
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