Glycosylation site analysis of human alpha‐1‐acid glycoprotein (AGP) by capillary liquid chromatography—electrospray mass spectrometry

Imre, Tı́mea; Schlosser, Gitta; Pocsfalvi, Gabriella; Siciliano, Rosa Anna; Molnár-Szöllősi, Éva; Kremmer, T.; Malorni, Antonio; Vékey, Károly

doi:10.1002/jms.938

Cited by 88 publications

(121 citation statements)

References 30 publications

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“…Trypsin cleavage may be blocked by the attached carbohydrates sterically when trypsin cleavage site was located right after glycosylated asparagine. 13 As a result, missed trypsin cleavage tends to occur more frequently when glycoprotein are digested. Moreover, some glycoproteins are not suitable for tryptic digestion either because they lack enough arginine and lysine residues in their sequences or they are not soluble at the optimal pH range of trypsin, for example, DQH sperm surface protein.…”

Section: Introductionmentioning

confidence: 99%

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

et al. 2009

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The study of protein glycosylation has lagged far behind the progress of current proteomics because of the enormous complexity, wide dynamic range distribution and low stoichiometric modification of glycoprotein. Solid phase extraction of tryptic N-glycopeptides by hydrazide chemistry is becoming a popular protocol for the analysis of N-glycoproteome. However, in silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. Herein, we describe a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion. The lysate of human liver tissue was digested with three proteases, that is, trypsin, pepsin and thermolysin, with different specificities, separately. Use of trypsin alone resulted in identification of 622 N-glycosites, while using pepsin and thermolysin resulted in identification of 317 additional N-glycosites. Among the 317 additional N-glycosites, 98 (30.9%) could not be identified by trypsin in theory because the corresponding in silico tryptic peptides are either too small or too big to detect in mass spectrometer. This study clearly demonstrated that the coverage of N-glycosites could be significantly increased due to the adoption of multiple enzyme digestion. A total number of 939 N-glycosites were identified confidently, covering 523 noredundant glycoproteins from human liver tissue, which leads to the establishment of the largest data set of glycoproteome from human liver up to now.

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Section: Introductionmentioning

confidence: 99%

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

et al. 2009

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“…In order to study glycoforms, the first step nearly always is enzymatic digestion. Often the sugar chain is cleaved off the peptide backbone, resulting in an oligosaccharide mixture [6][7][8][9]]. An alternative is to cleave the peptide chain using proteolytic enzymes (usually trypsin), which results in a peptide/glycopeptide mixture.…”

Section: ) Introductionmentioning

confidence: 99%

“…Glycopeptides retain information on both the peptide and the corresponding oligosaccharide chains. This approach started to gain importance in recent years [8,[10][11][12][13], and presents two major advantages: (1) Glycosylation sites and also glycosylation patterns at each site can be determined. (2) Protein-specific glycosylation patterns can be determined not only for pure glycoprotein samples, but for glycoprotein mixtures as well.…”

Section: ) Introductionmentioning

confidence: 99%

Fragmentation characteristics of glycopeptides

Vékey

Ozohanics

Tóth

et al. 2013

International Journal of Mass Spectrometry

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Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, "B" type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna-and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant.

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“…The RNase B glycopeptides did ionize in multiple charge states, and the spectra also contained peaks corresponding to missed tryptic cleavages of the glycopeptides (data not shown). The missed cleavages are likely the result of the glycosylation blocking the cleavage site, as described earlier [44], since several arginine and lysine residues are located very near the glycosylation site. See the amino acid sequence in Table 3.…”

Section: Quality Control Experiments 2-applicability To Different Glycmentioning

confidence: 70%

Label-free quantitation: A new glycoproteomics approach

Rebecchi

Wenke

et al. 2009

J. Am. Soc. Mass Spectrom.

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We demonstrate herein a method for quantifying glycosylation changes on glycoproteins. This novel method uses MS data of characterized glycopeptides to analyze glycosylation profiles, and several quality control tests were done to demonstrate that the method is reproducible, robust, applicable to different types of glycoproteins, and tolerant of instrumental variability during ionization of the analytes. This method is unique in that it is the first label-free quantitative method specifically designed for glycopeptide analysis. It can be used to monitor changes in glycosylation in a glycosylation site-specific manner on a single glycoprotein, or it can be used to quantify glycosylation in a glycoprotein mixture. During mixture analysis, the method can discriminate between changes in glycosylation of a given protein, and changes in the glycoprotein's concentration in the mixture. This method is useful for quantitative analyses in biochemical studies of glycoproteins, where changes in glycosylation composition can be linked to functional differences; it could also be implemented in the pharmaceutical industry, where glycosylation profiles of glycoprotein-based therapeutics must be quantified. Finally, quantification of glycopeptides is an important aspect of glycopeptide-based biomarker discovery, and our quantitative approach could be a valuable asset to this field as well, provided the compositions of the glycopeptides to be quantified are identifiable using other methods. (J Am Soc Mass

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Glycosylation site analysis of human alpha‐1‐acid glycoprotein (AGP) by capillary liquid chromatography—electrospray mass spectrometry

Cited by 88 publications

References 30 publications

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

Fragmentation characteristics of glycopeptides

Label-free quantitation: A new glycoproteomics approach

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