Back to Search
Start Over
High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS
- Source :
- Analytical Chemistry; January 2020, Vol. 92 Issue: 2 p1963-1971, 9p
- Publication Year :
- 2020
-
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. coliImmunity 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.
Details
- Language :
- English
- ISSN :
- 00032700 and 15206882
- Volume :
- 92
- Issue :
- 2
- Database :
- Supplemental Index
- Journal :
- Analytical Chemistry
- Publication Type :
- Periodical
- Accession number :
- ejs51781939
- Full Text :
- https://doi.org/10.1021/acs.analchem.9b04334