714961-01-0Relevant articles and documents
An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions
Ruan, Guihua,Wu, Zhenwei,Huang, Yipeng,Wei, Meiping,Su, Rihui,Du, Fuyou
, p. 54 - 60 (2016/04/20)
A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas.
The use of turbulent flow chromatography for rapid, on-line analysis of tryptic digests
Couchman,Jones,Moniz
, p. 2140 - 2146 (2015/10/28)
Rationale Following digestion of proteins with trypsin, digests are typically subjected to further 'clean-up' prior to liquid chromatography/mass spectometry (LC/MS) analysis, in order to reduce the complexity of the digested matrix, as well as helping to remove residual denaturants and reduction/alkylation reagents prior to injection onto the analytical HPLC column. Often, this is carried out using off-line techniques, and is not ideally suited to high-throughput workloads, for example in clinical laboratories. Methods Bovine serum albumin (BSA) was used as a model protein. Following denaturation with urea, reduction/alkylation, and digestion with trypsin, the analytical recovery of a selection of proteotypic BSA peptides was assessed using a two-dimensional, turbulent flow chromatography method. Peptides were identified using a Q Exactive mass spectometer operating in full-scan mode. Results Total analysis time (including the on-line sample clean-up) was 15 min per injection. Aside from the most hydrophilic peptide selected, ATEEQLK, recovery using the turbulent flow chromatography systems was greater than 30% for all remaining peptides (N=17), and exceeded 50% for 12 of the 18 peptides studied. There was a broad correlation between the hydrophobicity factor and the observed recovery. Conclusions This study suggests that turbulent flow chromatography offers a rapid, on-line alternative to solid-phase extraction for the analysis of peptide digests by LC/MS. A wide range of column chemistries are available, and the technique can be further optimised for analyses which are targetted to specific peptides. As with turbulent flow chromatography for small-molecule workflows, this approach may be ideally suited to high-throughput applications, such as those which are emerging from within clinical laboratories.
