Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing.
Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 mm × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 ng. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 μL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3-linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.
Van Andel Research Institute , Grand Rapids, Michigan 49503, United States.
Bryan S
BS
Reatini
Van Andel Research Institute , Grand Rapids, Michigan 49503, United States.
Elliot
E
Ensink
Bioprocessing Technology Institute , 138668, Singapore.
Brian
B
Liau
Van Andel Research Institute , Grand Rapids, Michigan 49503, United States.
Jessica Y
JY
Sinha
Medical University of South Carolina , Charleston, South Carolina 29425, United States.
Thomas W
TW
Powers
Van Andel Research Institute , Grand Rapids, Michigan 49503, United States.
Katie
K
Partyka
Protein Metrics, Inc. , San Carlos, California 94070, United States.
Marshall
M
Bern
University of Pittsburgh Medical Center , Pittsburgh, Pennsylvania 15237, United States.
Randall E
RE
Brand
Bioprocessing Technology Institute , 138668, Singapore.|National Institute for Bioprocessing Research and Training , Dublin, Ireland.
Pauline M
PM
Rudd
Protein Metrics, Inc. , San Carlos, California 94070, United States.
Doron
D
Kletter
Protein Metrics, Inc. , San Carlos, California 94070, United States.
Richard
R
Drake
Van Andel Research Institute , Grand Rapids, Michigan 49503, United States.
Brian B
BB
Haab
D000465:Algorithms; D006031:Glycosylation; D006801:Humans; D046228:Microarray Analysis; D055271:Mucin 5AC / Q000097:blood*; D011134:Polysaccharides / Q000032:analysis* / Q000138:chemical synthesis; D012984:Software
C524678:MUC5AC protein, human; D055271:Mucin 5AC; D011134:Polysaccharides