Structural Characterization of Glycoprotein Oligosaccharides and Glycosaminoglycans by Electrospray Linear Tandem Mass Spectrometry
Date Issued
2012
Date
2012
Author(s)
Chen, Shu-Ting
Abstract
In this study, negative linear ion trap mass spectrometry was used for structure characterization of oligosaccharides of glycoproteins and glycosaminoglycans. Glycosylation is one of the most abundant post-translational modification of protein. The oligosaccharides of glycoproteins play a critical role in terms of protein function. For structure characterization of oligosaccharide, our earlier study showed that a method based on p-aminobenzoic acid ethyl ester (ABEE) closed-ring labeling and negative ion ESI was presented. For larger oligosaccharides, to obtain linkages near the non-reducing end, a procedure involving alkaline degradation was introduced prior to ABEE labeling. This approach was time-consuming, so an effective method presented to provide linkage information near the non-reducing end is required. Oligosaccharides were labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) before negative MS2 analysis and the fragmentation occurred at the non-reducing end, so linkages near the terminus of oligosaccharides was obtained. The complementary information provided by ABEE and APTS-labeled oligosaccharides was utilized to elucidate the structure of larger oligosaccharides under negative MS2 analysis.
Prior the application of this combined approach, a method based on APTS labeling and negative ion ESI for linkage determination was investigated. The results revealed that the fragmentation of APTS labeling occurred primarily at the terminus. Therefore, linkage information starting at the non-reducing end was provided. In addition, the linkage assignments for terminal and internal linkage were based on different specific linkage fragment ions. Based on these ions, all the linkages of linear oligosaccharides could be determined from the non-reducing to the reducing terminus. Furthermore, the potential of the combined approach was demonstrated using larger neutral oligosaccharides, such as M5G2、M6G2、M8G2、M9G2 cleaved from ribonuclaease B. The results revealed that the linkage and branch assignments could be deduced from complementary information obtained from MS2 spectra of ABEE-labeled and APTS-labeled oligosaccharide. The linkages near the reducing end were derived from the spectrum of ABEE labeling, whereas linkages near the non-reducing end were assigned from the MS2 spectrum of APTS labeling.
Many glycoproteins appeared in living organisms contain abundant acidic oligosaccharides decorated with sialic acids. The potential of the combined approach was demonstrated using acidic oligosaccharides cleaved from transferrin and lactoferrin. To assure all negative charges were concentrated at the reducing terminus, a procedure involving methyl amidation was introduced prior to APTS labeling. The results showed that the specific linkage fragments for 2-3 and 2-6 linked sialic acids were obtained and the linkages near the non-reducing end could be determined. This combined approach for linkage assignments was used to deduce the linkages and branches of biantennary oligosaccharides cleaved from transferrin and lactoferrin. In addition, this approach was demonstrated with triantennary oligosaccharides (A3-A and A3-B glycans) cleaved from transferrin. The linkages near the reducing end were obtained from ABEE labeling, whereas linkages near the non-reducing end could be assigned based on a method involving methyl amidation, APTS labeling, and a short C18 packing probe separation coupled with negative ion ESI incorporating data-dependent tandem MS. Consequently, the linkages for A3-A and A3-B could be deduced.
For structure characterization of glycosaminoglycans, to successfully deduce the sulfation pattern of glycosaminoglycan, a strategy based on regioselective 6-O-desulfation reaction coupled with negative ESI analysis was developed. The information on sulfation pattern could be obtained based on glycosidic bond cleavages observed in the MS2 spectra of analytes before and after desulfation reaction. This strategy was demonstrated using a standard heparin and a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Additionally, a rougher estimation of the abundances of tetrasaccharides in shark cartilage used direct comparisons of relative peak areas.
Subjects
N-linked Oligosaccharides
APTS
ABE
Closed-Ring Labeling
Linkage analysis
Glycosaminoglycans
Shark cartilage chondroitin sulfate D
Type
thesis
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