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

José Marc Techner, Weston Kightlinger, Liang Lin, Jasmine Hershewe, Ashvita Ramesh, Matthew P. Delisa, Michael C. Jewett*, Milan Mrksich

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1963-1971
Number of pages9
JournalAnalytical Chemistry
Volume92
Issue number2
DOIs
StatePublished - Jan 21 2020

ASJC Scopus subject areas

  • Analytical Chemistry

Fingerprint Dive into the research topics of 'High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS'. Together they form a unique fingerprint.

  • Cite this