Characterization of an immobilized enzyme reactor for on-line protein digestion

Abstract

Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10 sec volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein’s molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15 hr.

Performance comparison of three trypsin columns used in liquid chromatography

“…This resulted in digestion/residential time of 5, 10, 48, and 480 s (8 min); respectively. The flow through fractions were collected and analyzed by LC MS(E) experiment as described previously [7]. The IMER digestions were compared to 15 h long in-solution digestion (Supplemental information, Tables S4A–C, and Fig.…”

“…Despite these shortcomings, IMERs have been shown to perform protein digestion within minutes [7,8], or even seconds [9–11]. Since the intermolecular collisions of trypsin are minimal, high enzyme concentration can be used in immobilized reactors, improving speed of digestion, while minimizing enzyme autolysis.…”

“…Preliminary digestion test of the prototype IMER with model protein was performed using Cytochrome C and BSA. Further evaluation of prototype IMER with more complex protein samples was described in a separate report [7]. …”

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

“…IM has also been coupled to CZE‐MS/MS or RPLC‐MS/MS for online protein digestion, peptide separation, and identification. However, IM has not played a significant role in routine deep bottom‐up proteomics studies.…”

“…Second, how well can IM perform for digestion of complex proteomes for deep proteomics compared with FT? Only a few reports in the literature applied the IM‐based fast protein digestion for large‐scale proteomics, resulting in 1000–3000 protein IDs from mammalian cell lines or tissues, and less than 1000 protein IDs from yeast cell lysate . The routine deep bottom‐up proteomics studies using FT digestion have approached over 8000 protein IDs from mammalian cell lines or tissues .…”