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

Abstract

High-throughput quantification of the posttranslational 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.

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

“… 10 , 11 Unfortunately, most of the MS methods are still relatively slow and reach only a moderate throughput, although positive development has been witnessed in recent years. 12 , 13 …”

Synthetic Glycobiology: Parts, Systems, and Applications

“…The modification of a sequon by a given ppGT is highly dependent on neighboring amino acids 80 and/or its structural context. 49 In fact, some ppGTs are dedicated to the modification of a single protein in their natural systems, 78 while others are more general. PpGTs with more relaxed specificities can be used to modify diverse target proteins by introducing an engineered sequence of amino acids known as a glycosylation tag (GlycTag), into the target protein sequence.…”

“… 122 The introduction of synthetic glycosylation pathways with NGTs as ppGTs results in the synthesis of diverse, minimal glycan motifs with applications in vaccines and therapeutics. 131 , 174 , 175 (b) The recapitulation and construction of glycosylation systems in cell-free platforms has enabled the in vitro expression of OSTs in nanodiscs, 237 the rigorous characterization of ppGT specificities, 49 , 80 the rapid discovery of new synthetic glycosylation pathways, 174 and the on-demand production of glycosylated therapeutics and vaccines by cell-free glycoprotein synthesis (CFGpS). 238 , 239 (c) Chemoenzymatic methods have been developed to install full-length human glycans.…”

“…The GlycoSCORES method has also been adapted to analyze intact glycoproteins, enabling the high-throughput synthesis and analysis of target protein variants with glycosylation sites at different positions. 49 …”

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

“…Specifically, by combining rapid in vitro biosynthesis of GTs, such as Ap NGT, in cell-free extract with high-throughput analysis of their activity using SAMDI-MS, the authors were able to systematically investigate the enzyme's substrate specificity using 3,480 unique peptides and 13,903 unique reaction conditions, revealing the optimal glycosylation sequon (Kightlinger et al, 2018 ). More recently, the same team extended the methodology to the analysis of intact glycoproteins (Techner et al, 2020 ), providing an exciting new avenue for the discovery and improvement of glycosylation enzymes. In addition, they used conditionally orthogonal peptide acceptor specificities of NGTs to site-specifically control installation of multiple distinct glycans (Lin et al, 2020 ).…”