In-Depth Structural Characterization of N-Linked Glycopeptides Using Complete Derivatization for Carboxyl Groups Followed by Positive- and Negative-Ion Tandem Mass Spectrometry

Abstract

Tandem mass spectrometry (MS/MS or MS(n)) is a powerful tool for characterizing N-linked glycopeptide structures. However, it is still difficult to obtain detailed structural information on the glycan moiety directly from glycopeptide ions. Here, we propose a new method for in-depth analysis of the glycopeptide structure using MS/MS. This method involves complete derivatization of carboxyl groups in glycopeptides. Methylamidation using PyAOP as a condensing reagent has been optimized for derivatizing all carboxyl groups in glycopeptides. By derivatizing carboxyl groups on the peptide moiety (i.e., Asp, Glu, and C-terminus), the glycopeptides efficiently produce informative glycan fragment ions, including the nonreducing end of the glycan moiety under negative-ion collision-induced dissociation (CID) conditions. These glycan fragment ions can define detailed structural features on the glycan moiety (e.g., the specific composition of the two antennae, the location of fucose residues, and the presence/absence of bisecting GlcNAc residues). For sialylated glycopeptides, carboxyl groups on sialic acid residues are simultaneously derivatized using methylamidation, suppressing preferential loss of residues during MS analysis. As a result, both sialylated and nonsialylated glycopeptides can be analyzed in the same manner. Positive-ion CID of methylamine-derivatized glycopeptides mainly provides information on peptide sequence and glycan composition, whereas negative-ion CID provides in-depth structural information on the glycan moiety. The derivatization step can be readily incorporated into conventional pretreatment for glycopeptide MS analysis without loss of sensitivity, making derivatization suitable for practical use.

Linkage-Specific Sialic Acid Derivatization for MALDI-TOF-MS Profiling of IgG Glycopeptides

“…[20][21][22] While these approaches have indeed shown improved sialylation analysis by MS, and can achieve recognition of sialic acid linkage upon fragmentation, no report has been made of a glycopeptide derivatization that reacts in a selective way to differently linked sialic acids.…”

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Mechanistic Studies and Methods for Improving the Structural Identification of Carbohydrates

“…Derivatization of carboxyl groups by methylamine using the condensing reagent (7-azabenzotriazol-1-yloxy)-tripyrrolidinophosphonium hexa uoro-phosphate (PyAOP) was applied by Nishikaze et al to conduct glycopro ling. 154) ey found that positive-ion CID of derivatized glycopeptides mainly provided information on peptide sequence and glycan composition, whereas negative-ion CID provided in-depth structural information on glycan moieties. PSD and CID were combined by Lattova et al to pro le derivatized N-linked glycans from ovalbumin.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…For example, ion mobility MS was used to separate and analyze intact ovomucoid glycopeptides (40) and urine glycopeptides from a patient diagnosed with Schindler disease (129). Furthermore, recent studies have explored the feasibility of measuring deprotonated glycopeptides in negative ion mode with (130) and without (131) derivatization. Innovative strategies for the derivatization of sialic acid residues have also been designed to achieve linkage-specific (␣2,3 versus ␣2,6-sialyl) information at the glycopeptide level (132) and to obtain more equal ionization response from neutral and sialylated glycopeptides (133).…”