Nanobiocatalysis for protein digestion in proteomic analysis

Abstract

The process of protein digestion is a critical step for successful protein identification in bottom-up proteomic analyses. To substitute the present practice of in-solution protein digestion, which is long, tedious, and difficult to automate, many efforts have been dedicated for the development of a rapid, recyclable and automated digestion system. Recent advances of nanobiocatalytic approaches have improved the performance of protein digestion by using various nanomaterials such as nanoporous materials, magnetic nanoparticles, and polymer nanofibers. Especially, the unprecedented success of trypsin stabilization in the form of trypsin-coated nanofibers, showing no activity decrease under repeated uses for 1 year and retaining good resistance to proteolysis, has demonstrated its great potential to be employed in the development of automated, high-throughput, and on-line digestion systems. This review discusses recent developments of nanobiocatalytic approaches for the improved performance of protein digestion in speed, detection sensitivity, recyclability, and trypsin stability. In addition, we also introduce approaches for protein digestion under unconventional energy input for protein denaturation and the development of microfluidic enzyme reactors that can benefit from recent successes of these nanobiocatalytic approaches.

Figure 1

Immobilization of trypsin in or on nanostructured materials such as nanoporous materials, nanoparticles, and nanofibers for efficient protein digestion.

Figure 2

Nanoporous materials as nanoscale reactors for protein digestion. (A) Trypsin adsorption was followed by the incubation in the protein solution. (B) Enrichment of proteins was followed by the incubation in the trypsin solution.

Figure 3

Schematic for the fabrication of enzyme coating on nanostructured materials.

Figure 4

Covalently-attached trypsin and trypsin coating on electrospun polymer nanofibers, and their SEM images (modified from reference [5]).

Figure 5

Strategies for protein digestion in proteomics using ‘unconventional’ energy inputs: (A) microwave irradiation, (B) high intensity focused ultrasound, and (C) pressure-assisted digestions.

Figure 6

(A) Pre-column immobilized enzyme reactor configuration. A protein is loaded in the reactor and digested to peptides, and then loaded in the analytical column. The valve is switched and a gradient elution is performed; peptides are separated, eluted, and detected by the mass spectrometry. (B) Post-column immobilized enzyme reactor configuration. (B1) A protein is retained in an analytical column, and then analyzed by mass spectrometry with no digestion. (B2) A protein is retained in an analytical column and digested in a post-column, and the peptides are analyzed by mass spectrometry. The protein from path B1 and its corresponding peptides from path B2 will have approximately the same retention time and elution profile enabling the correlation between them.