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

Anal Chem. 2020 Jan 21;92(2):1963-1971. doi: 10.1021/acs.analchem.9b04334. Epub 2020 Jan 7.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actinobacillus pleuropneumoniae / enzymology
  • Carrier Proteins / analysis*
  • Carrier Proteins / chemical synthesis
  • Carrier Proteins / genetics
  • Escherichia coli / chemistry
  • Escherichia coli Proteins / analysis*
  • Escherichia coli Proteins / chemical synthesis
  • Escherichia coli Proteins / genetics
  • 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*


  • Carrier Proteins
  • E colicin-binding immunity protein Im7, E coli
  • Escherichia coli Proteins
  • Glycoproteins
  • Peptide Library
  • Recombinant Proteins
  • Glycosyltransferases