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Anal Chem. 2020 Jan 7. doi: 10.1021/acs.analchem.9b04334. [Epub ahead of print]

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

Author information

1
Feinberg School of Medicine , Northwestern University , Chicago , Illinois 60611 , United States.
2
Department of Microbiology, Nancy E. and Peter C. Meinig School of Biomedical Engineering, Biochemistry, Molecular and Cell Biology, and Robert Frederick Smith School of Chemical and Biomolecular Engineering , Cornell University , Ithaca , New York 14853 , United States.

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.

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