Multi-Stage Mass Spectrometry Analysis of Sugar-Conjugated β-Turn Structures to be Used as Probes in Autoimmune Diseases

Abstract

Abstract. Synthetic sugar-modified peptides were identified as antigenic probes in the context of autoimmune diseases. The aim of this work is to provide a mechanistic study on the fragmentation of different glycosylated analogs of a synthetic antigenic probe able to detect antibodies in a subpopulation of multiple sclerosis patients. In particular the N-glucosylated type I′ β-turn peptide structure called CSF114(Glc) was used as a model to find signature fragmentations exploring the potential of multistage mass spectrometry by MALDI-LTQ Orbitrap. Here we compare the fragmentation of the glucosylated form of the synthetic peptide CSF114(Glc), bearing a glucose moiety on an asparagine residue, with less or non-immunoreactive forms, bearing different sugar-modifications, such as CSF114(GlcNAc), modified with a residue of N-acetylglucosamine, and CSF114[Lys 7 (1-deoxyfructopyranosyl)], this last one modified with a 1-deoxyfructopyranosyl moiety on a lysine at position 7. The analysis was set up using a synthetic compound specifically deuterated on the C-1 to compare its fragmentation with the fragmentation of the undeuterated form, and thus ascertain with confidence the presence on an Asn(Glc) within a peptide sequence. At the end of the study, our analysis led to the identification of signature neutral losses inside the sugar moieties to characterize the different types of glycosylation/glycation. The interest of this study lies in the possibility of applyimg this approach to the discovery of biomarkers and in the diagnosis of autoimmune diseases.

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

“…In this approach, the glycopeptide ion is selected and fragmented resulting in a variety of fragment ions predominantly due to the cleavage of glycosidic linkages. The peptide ion carrying a single HexNAc, is subjected to a second ion isolation/fragmentation cycle, resulting in fragmentation of the peptide moiety [25–28]. However, this also requires prior knowledge of the targeted peptides to select for MS 3 , thus limiting its throughput [25–28].…”

“…The peptide ion carrying a single HexNAc, is subjected to a second ion isolation/fragmentation cycle, resulting in fragmentation of the peptide moiety [25–28]. However, this also requires prior knowledge of the targeted peptides to select for MS 3 , thus limiting its throughput [25–28]. Several other reports alternate collision energies and collect sequential MS/MS on the same precursor, based on the observation that lower energy preferentially fragment glycans and higher energy can fragment peptide backbones [29, 30].…”