Separation and identification of isomeric glycopeptides by high field asymmetric waveform ion mobility spectrometry

Abstract

The analysis of intact glycopeptides by mass spectrometry is challenging due to the numerous possibilities for isomerization, both within the attached glycan and the location of the modification on the peptide backbone. Here, we demonstrate that high field asymmetric wave ion mobility spectrometry (FAIMS), also known as differential ion mobility, is able to separate isomeric O-linked glycopeptides that have identical sequences but differing sites of glycosylation. Two glycopeptides from the glycoprotein mucin 5AC, GT(GalNAc)TPSPVPTTSTTSAP and GTTPSPVPTTST(GalNAc)TSAP (where GalNAc is O-linked N-acetylgalactosamine), were shown to coelute following reversed-phase liquid chromatography. However, FAIMS analysis of the glycopeptides revealed that the compensation voltage ranges in which the peptides were transmitted differed. Thus, it is possible at certain compensation voltages to completely separate the glycopeptides. Separation of the glycopeptides was confirmed by unique reporter ions produced by supplemental activation electron transfer dissociation mass spectrometry. These fragments also enable localization of the site of glycosylation. The results suggest that glycan position plays a key role in determining gas-phase glycopeptide structure and have implications for the application of FAIMS in glycoproteomics.

Figure 1

The extracted ion chromatograms for the doubly charged precursor ion m/z 852.91 (black), fragment c₁₀ (red) from the glycopeptide GTTPSPVPTTSTTSAP, and fragment z₁₀ (blue) from the glycopeptide GTTPSPVPTTSTTSAP. T represents the threonine amino acid residue modified with N-acetylgalactosamine, GalNAc.

Figure 2

(a) Supplemental activation ETD mass spectrum of [M + 2H]²⁺ ions of peptide A, recorded at a CV of −23.7 V. (b) Supplemental activation ETD mass spectrum of [M + 2H]²⁺ ions of peptide B, recorded at a CV of −25.5 V. The CV values represent the maximum transmission of the precursor ions through the FAIMS device.

Figure 3

(a) Extracted ion chromatograms for the reporter c and z ions of peptide A (c₈, c₉, c₁₀, c₁₁, and c₁₂, red) and peptide B (c₈, c₉, c₁₀, c₁₁, c₁₂, z₁₀, and z₁₂, blue). (b) Supplemental activation ETD mass spectrum recorded at a CV of −27.6 V. Reporter ions for peptide B are labeled in blue. (c) Supplemental activation ETD mass spectrum recorded at a CV of −20.7 V. Reporter ions for peptide A are labeled in red.