1. High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS.
- Author
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Techner JM, Kightlinger W, Lin L, Hershewe J, Ramesh A, DeLisa MP, Jewett MC, and Mrksich M
- Subjects
- Actinobacillus pleuropneumoniae enzymology, Carrier Proteins chemical synthesis, Carrier Proteins genetics, Escherichia coli chemistry, Escherichia coli Proteins chemical synthesis, Escherichia coli Proteins genetics, Glycoproteins chemical synthesis, Glycoproteins genetics, Glycosylation, Glycosyltransferases chemistry, Mutation, Peptide Library, Proof of Concept Study, Recombinant Proteins analysis, Recombinant Proteins chemical synthesis, Recombinant Proteins genetics, Carrier Proteins analysis, Escherichia coli Proteins analysis, Glycoproteins analysis, High-Throughput Screening Assays methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- 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.
- Published
- 2020
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