Direct structural assignment of neutral and sialylated N‐glycans of glycopeptides using collision‐induced dissociation MSⁿ spectral matching

Abstract

Mass spectrometry analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS n spectra acquired by ESI-Linear IT TOF MS in the positive-and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS 2 spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcβ1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na] 2+ precursor ion are shared with both of the resulting fragments. These fragments are sodiated B n -type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagines (N) residue. The structure of N-glycan was assigned by comparing MS 3 spectra derived from both the sodiated B n -type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS n spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS n spectra of deprotonated B n -type fragment ions of N-glycopeptide and the PA N-glycan standard.The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These -2 -results suggest that the approach based on MS n spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.

Addressing the glycan complexity by using mass spectrometry: In the pursuit of decoding glycologic

“…The structurally isomeric glycans produce very similar spectra consisting of a series of ions with the same m/z values. However, the heights of some of the signals are often different, making it possible to distinguish the isomeric compounds by comparing the spectra (Figure 4) [51,52]. The spectral matching method relies on the fact that the different chemical bonds in isomeric compounds require different energies to dissociate, producing a distinguishable pair of spectra.…”

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

“…So far, a greater production of di erent fragment ions has been reported than in positive-ion mode. 42,[54][55][56][57] We performed CID experiments using a large set of glycopeptides in an attempt to understand the fragmentation behavior of deprotonated glycopeptides. 58) e obtained results indicate that the detectable fragment ion species are variable, strongly dependent on their amino acid composition and sequence, and therefore assignment of the CID spectra becomes somewhat complicated.…”

“…8, negative-ion CID of a test glycopeptide (desialylated sialoglycopeptide, dSGP) resulted in abundant formation of peptide fragments (e.g., 0,2 X 0 +peptide and peptide−NH 3 ) with weak signals of type (i) glycan fragments (e.g., 2,4 A R and 2,4 A R-1 ). MS 3 spectra of the glycan fragments may provide additional information on the glycan structure, 42,54,55) but it is ine cient because of weak signals. Derivatizing the C-terminal carboxyl group on the peptide moiety greatly enhanced the production of glycan fragments (Fig.…”

Glycoscience aids in biomarker discovery

“…By combining the compositional information provided by MS analysis with biological knowledge of probable glycan structures, glycan structures may be inferred (37). If available, tandem MS can also be applied to further elucidate the glycan structure (2,14,26,(34)(35)(36)(38)(39)(40)(41)(42). Glycan compositional mass profiling is an attractive option for biomarker discovery due to the simplicity of the data analysis and its potential for rapid-and high-throughput applications (when done by MALDI-MS).…”