Separation and detection of minimal length glycopeptide neoantigen epitopes centering the GSTA region of MUC1 by liquid chromatography/mass spectrometry.

Abstract

We previously isolated antibodies binding to glycopeptide neoantigen epitopes centering the GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering the GSTA sequence are also predicted by NetMHC programs to bind to MHC molecules. Detecting MUC1 glycopeptide epitopes remains a challenge since antigenic epitopes are often shorter than 10 amino acids. In this study, we used pronase from Streptomyces griseus, which has no amino acid sequence preference for enzymatic cleavage sites, to digest synthetic glycopeptides RPAPGST (Tn)APPAHG and RPAPGS (Tn)TAPPAHG, and analyzed the digests by liquid chromatography/mass spectrometry (LC/MS) using electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) methods with an Orbitrap Fusion Lumos Tribid mass spectrometer. We found that short glycopeptides containing 8 to 11 amino acids could be efficiently generated by pronase digestion. Such glycopeptides of minimal epitope lengths were clearly distinguished by characteristic MS/MS ion patterns and LC elution profiles. A glycopeptide library was generated which may serve as a standard for measuring neoantigen epitopes centering the GSTA sequence. Our data established the LC/MS/MS identities of a clinically relevant MUC1 glycopeptide neoantigen epitope centering the GSTA motif. A library of short MUC1 glycopeptides centered on the GSTA motif was created, which is a critical step for analysis of such antigen epitopes in real biological samples.