Site-specific N-glycosylation characterization of micro monoclonal immunoglobulins based on EThcD-sceHCD-MS/MS

Front Immunol. 2022 Sep 15;13:1013990. doi: 10.3389/fimmu.2022.1013990. eCollection 2022.

Abstract

Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma and monoclonal gammopathy of renal significance. Because of the complicated purification method and the low stoichiometry of purified protein and glycans, site-specific N-glycosylation characterization for monoclonal immunoglobulin is still challenging. To profile the site-specific N-glycosylation of monoclonal immunoglobulins is of great interest. Therefore, in this study, we presented an integrated workflow for micro monoclonal IgA and IgG purification from patients with multiple myeloma in the HYDRASYS system, in-agarose-gel digestion, LC-MS/MS analysis without intact N-glycopeptide enrichment, and compared the identification performance of different mass spectrometry dissociation methods (EThcD-sceHCD, sceHCD, EThcD and sceHCD-pd-ETD). The results showed that EThcD-sceHCD was a better choice for site-specific N-glycosylation characterization of micro in-agarose-gel immunoglobulins (~2 μg) because it can cover more unique intact N-glycopeptides (37 and 50 intact N-glycopeptides from IgA1 and IgG2, respectively) and provide more high-quality spectra than sceHCD, EThcD and sceHCD-pd-ETD. We demonstrated the benefits of the alternative strategy in site-specific N-glycosylation characterizing micro monoclonal immunoglobulins obtained from bands separated by electrophoresis. This work could promote the development of clinical N-glycoproteomics and related immunology.

Keywords: EThcD-sceHCD; N-glycosylation; mass spectrometry; micro; monoclonal immunoglobulin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid / methods
  • Glycopeptides
  • Glycosylation
  • Humans
  • Immunoglobulin A
  • Immunoglobulin G
  • Multiple Myeloma*
  • Polysaccharides
  • Sepharose
  • Tandem Mass Spectrometry* / methods

Substances

  • Glycopeptides
  • Immunoglobulin A
  • Immunoglobulin G
  • Polysaccharides
  • Sepharose