Novel Isobaric Tagging Reagent Enabled Multiplex Quantitative Glycoproteomics via Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry

Abstract

Protein glycosylation, covalent attachment of carbohydrates to polypeptide chains, is a highly important post-translational modification involved in many essential physiological processes. Comprehensive site-specific and quantitative analysis is crucial for revealing the diverse functions and dynamics of glycosylation. To characterize intact glycopeptides, mass spectrometry (MS)-based glycoproteomics employs versatile fragmentation methods, among which electron-transfer/higher-energy collision dissociation (EThcD) has gained great popularity. However, the inherent limitation of EThcD in fragmenting low-charge ions has prevented its widespread applications. Furthermore, there is a need to develop a high-throughput strategy for comparative glycoproteomics with a large cohort of samples. Herein, we developed isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) tags to increase the charge states of glycopeptides, thereby improving the fragmentation efficiency and allowing for in-depth intact glycopeptide analysis, especially for sialoglycopeptides. Moreover, the unique reporter ions of DiLeuEN-labeled glycopeptides generated in tandem MS spectra enable relative quantification of up to four samples in a single analysis, which represents a new high-throughput method for quantitative glycoproteomics.

Keywords: DiLeuEN; EThcD; isobaric labeling; mass spectrometry; quantitative glycoproteomics.

Figure 1.

(A) Structure and synthetic routes of DiLeuEN tag; (B) Isotope configurations of 4-plex DiLeuEN tags (purple, red, and green dots represent ¹³C, ¹⁵N, and ¹⁸O isotope position respectively).

Figure 2.

(A-C) MALDI-MS profiling of native, dimethylated and DiLeuEN labeled SGP; (D-F) ESI-MS profiling of native, dimethylated and DiLeuEN labeled SGP.

Figure 3.

Representative EThcD-MS/MS spectra for the sialoglycopeptides from bovine fetuin. (A) underivatized, (B) DiLeuEN derivatized. (To emphasize ETD fragmentation improvement, only c/z ions are highlighted, whereas b/y ions and glycan fragments are not annotated)

Figure 4.

(A) Box plot of the relative quantification performance of 4-plex DiLeuEN labeled tryptic digest of bovine fetuin; (B) The comparison of the number of intact glycopeptides, glycosylation sites and glycoproteins between native and DiLeuEN labeled tryptic digest samples of Panc1 cell; (C) Volcano plot showing the quantification result of tryptic digest peptides of Panc1 cell incubated in 0.1% FA versus lyophilized control (37 sialylglcopeptides decayed, green dots); (D) Representative EThcD-MS/MS spectra for the 4-plex DiLeuEN labeled glycopeptide from Panc1 cell.