Abstract
The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, grycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3684-3692 |
| Number of pages | 9 |
| Journal | Analytical Chemistry |
| Volume | 80 |
| Issue number | 10 |
| DOIs | |
| State | Published - May 15 2008 |
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ASJC Scopus subject areas
- Analytical Chemistry
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Factors that influence fragmentation behavior of N-linked glycopeptide ions. / Seipert, Richard R.; Dodds, Eric D.; Clowers, Brian H.; Beecroft, Sean M.; German, J. Bruce; Lebrilla, Carlito B.
In: Analytical Chemistry, Vol. 80, No. 10, 15.05.2008, p. 3684-3692.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Factors that influence fragmentation behavior of N-linked glycopeptide ions
AU - Seipert, Richard R.
AU - Dodds, Eric D.
AU - Clowers, Brian H.
AU - Beecroft, Sean M.
AU - German, J. Bruce
AU - Lebrilla, Carlito B
PY - 2008/5/15
Y1 - 2008/5/15
N2 - The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, grycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.
AB - The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, grycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.
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U2 - 10.1021/ac800067y
DO - 10.1021/ac800067y
M3 - Article
C2 - 18363335
AN - SCOPUS:43949107914
VL - 80
SP - 3684
EP - 3692
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 10
ER -