Immobilization of trypsin onto porous methacrylate-based monolith for flow-through protein digestion and its potential application to chiral separation using liquid chromatography

Abstract

Monolithic columns for analytical applications have attracted the researcher's attention. In this work, the laboratory-made organic-polymer monolithic column is modified with trypsin and further applied as a nanobiocatalyst microreactor and a stationary phase for separating chiral compounds by liquid chromatography. The monolith was synthesized by in-situ copolymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or trimethylolpropane trimethacrylate (TRIM) as a crosslinking agent, with porogen of 1,4-butanediol/propanol/water (4:7:1 v/v) and AIBN as the radical polymerization initiator inside PEEK and silicosteel tubings (1.0 mm i.d  100 mm) at 60 C for 12 h. A total monomer ratio (%T) and crosslinking agent (%C) of 40:25 and 28:12 were applied to prepare poly-(GMA-co-EDMA) and poly-(GMA-co-TRIM), respectively. The produced monoliths were further modified by introducing trypsin (10 mg/L) through the ring-opening reaction of the epoxide group existing in the monolithic column. The trypsin-immobilized poly-(GMA-co-EDMA) monolithic column was applied as the nanobiocatalyst microreactor for online/flow-through and rapid digestion of β-casein sample into its peptide fragments. The trypsin-immobilized poly-(GMA-co-TRIM) column has potential application to be used as the HPLC stationary phase for the separation of R/S-citronellal enantiomers.

Thiol-ene-based microfluidic chips for glycopeptide enrichment and online digestion of inflammation-related proteins osteopontin and immunoglobulin G

“…Protein digestion using a proteolytic enzyme is an unavoidable step involved in these methods, facilitating the identification through analysis of smaller peptides. In order to reduce the sample volume needed for the analysis of biological samples, and to increase the time-to-result efficiency, microfluidic devices with immobilized enzymes providing online digestion have been developed [14][15][16][17][18][19][20][21][22][23][24][25]. Microfluidic devices with integrated electrospray emitters, or prepared in fused silica capillary, or similar microfabricated devices could also be coupled to ESI-MS for online detection [23,24,[26][27][28][29].…”

Current trends in the development of polymer‐based monolithic stationary phases

“…However, due to many chiral centres, trypsin can also act as a chiral selector 66 . Amalia et al 67 . prepared a poly(GMA‐ co ‐trimethylolpropane trimethacrylate) monolithic stationary phase with immobilized trypsin and used it to separate R/S citronellal enantiomers.…”

“…However, due to many chiral centres, trypsin can also act as a chiral selector. 66 Amalia et al 67 prepared a poly(GMAco-trimethylolpropane trimethacrylate) monolithic stationary phase with immobilized trypsin and used it to separate R/S citronellal enantiomers. Although baseline resolution was not achieved, it was possible to identify split peaks with different retention times.…”

“…Trypsin‐immobilized poly (GMA‐ co ‐EDMA) monolithic micro reactor for online digestion of β‐casein was prepared by Amalia et al 67 . The effect of the digestion flow rate and the temperature of digestion were investigated to obtain the optimum interaction between the protein and the immobilized trypsin.…”

“…118 Recently, several reviews dealing with the preparation of enzymatic reactors based on polymer monoliths have been published. [119][120][121] Trypsin-immobilized poly (GMA-co-EDMA) monolithic micro reactor for online digestion of β-casein was prepared by Amalia et al 67 The effect of the digestion flow rate and the temperature of digestion were investigated to obtain the optimum interaction between the protein and the immobilized trypsin. The number of appeared peaks, as well as the peak intensities at the digestion flow rate of 1 μL/min (residence time 80 min) was higher than that in a flow rate of 5 μL/min (residence time 16 min) and 10 μL/min (residence time 8 min).…”