Enhanced Detection and Identification of Glycopeptides in Negative Ion Mode Mass Spectrometry

Abstract

A combined MS and MS/MS approach implemented with MALDI FT-ICR MS in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode mass spectrometry (MS) analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed towards enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to the positive ion mode MS/MS analysis.

Challenges in Separations of Proteins and Small Biomolecules and the Role of Modern Mass Spectroscopy Tools for Solving Them, as Well as Bypassing Them, in Structural Analytical Studies of Complex Biomolecular Mixtures

“…Aside from the analytical problems related to the separation and analysis of glycans, the additional challenges in analysis of glycopeptides are: (i) presence of the peptide backbone, (ii) identification of glycopeptides (typically mixed with peptides), (iii) identification of glycosylation sites, (iv) identification of glycan structures, and (v) characterization of microheterogeneity of glycosylation. Negative ion mode MALDI MS/MS has been successfully applied for analysis of N-glycopeptides [78]. MALDI MS allows detailed MS/MS characterization of small biomolecules, but it lacks efficient separation of isomers capability.…”

“…The biggest drawbacks of LC-MS analyses of released N-glycans are twofold: (i) loss of information about N-glycosylation sites on proteins from which the N-glycans were released; and (ii) loss of information about N-glycosylation microheterogeneity patterns on N-glycosylation sites on respective proteins. This information can be obtained using MALDI MS/MS approach, as shown in Figure 17, on example of bovine lactoferrin [78]. Such glycoprotein samples are extremely complex to analyze using MS, especially when present in complex glycoproteomic sample mixtures.…”

“…Circles (green and yellow), squares (blue), and triangles (red) represent hexose, GlcNAc, and fucose residues, respectively. Reproduced with permission from [78]. Copyright American Chemical Society, 2010.…”

“…Two main MS methods have been successfully employed for structural analyses of such mixtures: (i) MALDI MS and (ii) nanoESI-LC-MS. Although more limited compared to LC-MS, MALDI MS has been shown to be capable of analyzing very complex mixtures of small biomolecules, such as glycopeptides [78] and lipid extracts [3]. While the data analysis part of these analyses is inherently more complex, especially when isomeric structures are present in the sample, they do not require an extensive set of bioinformatic tools which LC-MS analyses rely on.…”

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…Actually, Nwosu et al reported on the enhanced detection of relatively short glycopeptides in the negative-ion mode, compared to the positive-ion mode. 53) Despite its potential usefulness in detecting glycopeptide signals, little information is available in terms of our understanding of the fragmentation of deprotonated glycopeptides. So far, a greater production of di erent fragment ions has been reported than in positive-ion mode.…”