Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin fluorescence detection.
Protein glycosylation has been implicated in key biological processes including immunological recognition, cellular adhesion, protein folding, and signaling as well as disease progression. Although several methods are available to assess glycosylation of protein structures, none of them is able to screen complex biological samples at a global as well as an individual scale. A novel strategy presented here uses an all-liquid phase enrichment and prefractionation methodology coupled to glycoprotein microarray technology using a multiple lectin-based, biotin-streptavidin detection scheme. Selective detection of glycan structures was made possible by employing multiple lectins to screen glycoprotein standards as well as serum samples from normal subjects or patients with chronic pancreatitis or pancreatic cancer. Interestingly, in some instances, a greater degree of glycosylation was seen in proteins that were underexpressed based on the reversed-phase chromatogram alone. Studies with standard proteins established the limits of detection to be in the 2.5-5-fmol range. Studies on serum samples showed differences in glycosylation patterns, particularly with respect to sialylation, mannosylation, and fucosylation, in normal, pancreatitis, and cancer sera. By coupling glycoprotein enrichment and fractionation with a microarray platform, we have shown that naturally occurring glycoproteins from human serum can be screened and characterized for different glycan structures, thereby allowing one to do comparative studies that monitor individual glycosylation changes within a glycoproteome representing different biological states. This approach may be useful to identify potential biomarkers in cancer.
Patwa
Tasneem H
TH
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Zhao
Jia
J
Anderson
Michelle A
MA
Simeone
Diane M
DM
Lubman
David M
DM
D015415:Biomarkers / Q000097:blood; D002853:Chromatography, Liquid; D005260:Female; D005453:Fluorescence; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry; D008297:Male; D010182:Pancreatic Diseases / Q000097:blood*; D040081:Protein Array Analysis / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D013054:Spectrophotometry, Atomic; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D006023:Glycoproteins; D037102:Lectins