Characterization of protein glycosylation using chip-based infusion nanoelectrospray linear ion trap tandem mass spectrometry.
Mass spectrometry (MS) has the potential to revolutionize structural glycobiology and help in the understanding of how post-translation events such as glycosylation affect protein activities. Several approaches to determine the structure of glycopeptides have been used successfully including fast atom bombardment, matrix-assisted laser desorption ionization, and electrospray ionization with a wide variety of mass analyzers. However, the identification of glycopeptides in a complex mixture still remains a challenge. The source of this challenge is primarily due to the poor ionization efficiency and rapid degradation of glycopeptides. In this report we describe the use of a chip-based infusion nanoelectrospray ionization technique in combination with a recently developed linear ion trap for identification and characterization of glycosylation in complex mixtures. Two standard synthetic glycans were analyzed using multiple-stage fragmentation analysis in both positive and negative ionization modes. In addition, the high mannose type N-glycosylation in ribonuclease B (RNase B) was used to map the glycosylation site and obtain the glycan structures. We were able to map the glycosylation site and obtain the glycan structures in RNase B in a single analysis. The results reported here demonstrate that the fully automated chip-based nanoelectrospray linear ion trap platform is a valuable system for oligosaccharide analyses due to the unique MS/MS and MS(n) capability of the linear ion trap and the extended analysis time provided by the ionization technique.
Zhang
Sheng
S
Advion BioSciences, Inc. Ithaca, NY, USA.
Chelius
Dirk
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D006020:Glycopeptides; D006027:Glycosides; D010446:Peptide Fragments; D011134:Polysaccharides; D012260:Ribonucleases; C029600:ribonuclease B