Complementary structural information of positive‐ and negative‐ion MSⁿ spectra of glycopeptides with neutral and sialylated N‐glycans

Abstract

Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n = 2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N-acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1 Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1-type cross-ring cleavage (83.1 Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6-type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6-type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary.

Liquid Chromatography/Mass Spectrometry (LC/MS)-Based Glycoproteomics Technologies for Cancer Biomarker Discovery

“…3). It has successfully determined the three glycosylated sites and the glycoform structures in Thy-1, a glycosylphosphatidylinositol-anchorred protein (67), and determined the structures of neutral and sialylated N-glycans attached to chicken egg yolk glycopeptides (69). Thus, the MS n technology will be a powerful tool for glycoproteome analysis after the collection speed of MS n spectra is increased to the LC-MS time scale.…”

“…High-speed multi-stage MS/MS technology, or MS n , has emerged as a technology that enables both peptide assignment and structural determination of posttranslational modifications, such as the site of glycosylation and the attached glycan structure (65)(66)(67)(68)(69)(70). The method determines the structure by collecting information from a series of fragment ions generated by multiple CID processes, typically three times (MS 3 ; Fig.…”

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.…”

Structural assignment of disialylated biantennary N‐glycan isomers derivatized with 2‐aminopyridine using negative‐ion multistage tandem mass spectral matching

“…42 200.4, respectively, by using an a2-3-sialyltransferase reaction at 378C for 12 h in 50 mM HEPES buffer (pH 7.0) containing 10 mM MnCl 2 , 0.1% Triton CF-54, 0.1% NaN 3 , 600 mM CMP-Neu5Ac, and 2 mU a2-3-sialyltransferase. Finally, each of them was purified again with the Develosil C30-UG column.…”