Separation and detection of minimal length isomeric glycopeptide neoantigen epitopes centering GSTA region of MUC1 by LC-MS

MUC1 ranks No.2 on the list of targets for cancer immunotherapy. We previously reported monoclonal antibodies binding to glycopeptide neoantigen epitopes centering GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering GSTA sequence are also predicted by NetMHC pro...

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Bibliographic Details
Published inbioRxiv
Main Authors Zhou, Dapeng, Xiao, Kaijie, Tian, Zhixin
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 24.04.2019
Subjects
Amino acid sequence
Amino acids
Cancer
Cancer immunotherapy
Chromatography
Clostripain
Electron transfer
Endopeptidase
Epitopes
Immunotherapy
Isomers
Liquid chromatography
Major histocompatibility complex
Mass spectroscopy
Monoclonal antibodies
Neoantigens
Nucleotide sequence
Pronase
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Summary:MUC1 ranks No.2 on the list of targets for cancer immunotherapy. We previously reported monoclonal antibodies binding to glycopeptide neoantigen epitopes centering GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering GSTA sequence are also predicted by NetMHC programs to bind to MHC molecules, although empirical data are lacking. Detecting isomeric MUC1 glycopeptide epitopes by mass spectrometry (MS) remains a technical challenge since antigenic epitopes are often shorter than 10 amino acids. MUC1 digests by Arg-C-specific endopeptidase clostripain could generate heterogenous icosapeptides, but isomeric 20-residue glycopetides could not be separated by liquid chromatography. In this study, we used pronase from Streptomyces griseus, which has no amino acid sequence preference for enzymatic cleavage sites, to digest a pair of synthetic glycopeptide isomers RPAPGST(Tn)APPAHG and RPAPGS(Tn)TAPPAHG, and analyzed the digests by LC-MS using electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) methods. The results showed that glycopeptide isomers containing 8 to 11 amino acids could be efficiently generated by pronase digestion. Such glycopeptide isomers of minimal epitope lengths were clearly distinguished by characteristic MS/MS ion patterns and elution profiles of liquid chromatography. A glycopeptide library was generated which may serve as standards for measuring neoantigen epitopes centering GSTA sequence.
DOI:10.1101/617308