Application of StrucGP in medical immunology:site-specific N-glycoproteomic analysis of macrophages

The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals.Meanwhile,the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions.In this work,we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis.The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry(MS)-based glycoproteomic approaches,followed by the large-scale mapping of site-specific glycan structures via StrucGP.Results revealed that bisected GlcNAc,core fucosylated,and sialylated glycans(e.g.,HexNAc4Hex5Fuc1Neu5Ac1,N4H5F1S1)were increased in M1 and M2 macrophages,especially in the latter.The findings indicated that these structures may be closely related to macrophage polarization.In addition,a high level of glycosylated PD-L1 was observed in M1 macrophages,and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1,and Asn-200 contained Lewis epitopes.The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.

Proteomic characterization of four subtypes of M2 macrophages derived from human THP-1 cells

Macrophages are widely distributed immune cells that contribute to tissue homeostasis.Human THP-1 cells have been widely used in various macrophage-associated studies,especially those involving pro-inflammatory M1 and anti-inflammatory M2 phenotypes.However,the molecular characterization of four M2 subtypes(M2a,M2b,M2c,and M2d)derived from THP-1has not been fully investigated.In this study,we systematically analyzed the protein expression profiles of human THP-1-derived macrophages(M0,M1,M2a,M2b,M2c,and M2d)using quantitative proteomics approaches.The commonly and specially regulated proteins of the four M2 subtypes and their potential biological functions were further investigated.The results showed that M2a and M2b,and M2c and M2d have very similar protein expression profiles.These data could serve as an important resource for studies of macrophages using THP-1 cells,and provide a reference to distinguish different M2 subtypes in macrophage-associated diseases for subsequent clinical research.