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Title: High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS.

Authors: Techner, José-Marc; Kightlinger, Weston; Lin, Liang; Hershewe, Jasmine; Ramesh, Ashvita; DeLisa, Matthew P; Jewett, Michael C; Mrksich, Milan

Published In Anal Chem, (2020 01 21)

Abstract: High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

PubMed ID: 31854989 Exiting the NIEHS site

MeSH Terms: Actinobacillus pleuropneumoniae/enzymology; Carrier Proteins/analysis*; Carrier Proteins/chemical synthesis; Carrier Proteins/genetics; Escherichia coli Proteins/analysis*; Escherichia coli Proteins/chemical synthesis; Escherichia coli Proteins/genetics; Escherichia coli/chemistry; Glycoproteins/analysis*; Glycoproteins/chemical synthesis; Glycoproteins/genetics; Glycosylation; Glycosyltransferases/chemistry; High-Throughput Screening Assays/methods*; Mutation; Peptide Library; Proof of Concept Study; Recombinant Proteins/analysis; Recombinant Proteins/chemical synthesis; Recombinant Proteins/genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods*

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