Enzyme‐immobilized reactors for rapid and efficient sample preparation in MS‐based proteomic studies

Abstract

Proteolysis is a key step in proteomic studies integrated with MS analysis but the conventional method of in-solution digestion is limited by time-consuming procedures and low sensitivity. Furthermore, obtaining reliable peptide maps and meaningful sequence data using MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions. Recently, various immobilized-enzyme reactors have been developed for highly efficient proteolysis in MS-based proteomic analysis. This review focuses on the proteolysis step using protease-immobilized reactors and rapid analysis of protein sequences. We describe the preparation of enzyme reactors by several techniques and protein digestion under unusual conditions. Analysis of posttranslational modifications by enzyme reactors prepared using our immobilization method is presented as a model application. Analysis systems using immobilized-enzyme reactors are expected to become useful tools for proteomic studies and diverse applications in biotechnology.

Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion

“…This method adopted an ultrafast trypsin digestion protocol we developed for assays for glycan analysis 44 and site-specific oxidation, 45 and avoided the assay artifacts of oxidation, deamidation, and isomerization induced during the cLC-MS method. 8,[34][35][36][37]39 With uLC-MS available for comparison, assay artifacts for Asn deamidation and Asp isomerization at certain sites were clearly shown in the longer digestion procedure. For example, deamidation at Asn316 of mAb A was »120-fold overestimated by an 18-hour digestion in the cLC-MS compared to the 5-min digestion procedure (60% vs 0.5%).…”

“…[31][32][33] The treated proteins are digested with trypsin or other proteases, and the peptides generated are separated by RP-HPLC for MS. The sample digestion step for the method is a time consuming process that could take up to 24 hours at 37 C, a step that could potentially induce artifacts 34 such as asparagine deamidation, 8,[35][36][37] N-terminal glutamine cyclization, 38 and methionine oxidation. 39,40 In addition, the long preparation time hampers the high throughput analysis for a large number of samples in forced degradation studies and clone and media selection.…”

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

“…Thus, another goal is to develop procedures that accelerate the enzyme reaction step: one approach is the use of immobilized enzyme reactors (IMERs), as they have several advantages compared to standard in-solution/in-gel approaches, such as larger enzyme to substrate ratio, higher digestion efficiency, long-term stability and reusability [29], [30].…”

Recent progress in nanobiocatalysis for enzyme immobilization and its application

“…In addition, in solutiondigestion often induces asparagine deamination, methionine oxidation, and N-terminal glutamine cyclization on target proteins owing to the elevated temperature and alkaline pH during the digestion. Accordingly, developing a novel digestion method is the most important topic in proteomics coupled with MS analysis [75][76][77]. As an alternate method, many researchers have designed an immobilized-protease reactor.…”