Dual Matrix-Based Immobilized Trypsin for Complementary Proteolytic Digestion and Fast Proteomics Analysis with Higher Protein Sequence Coverage

Abstract

In an age of whole-genome analysis, the mass spectrometry-based bottom-up strategy is now considered to be the most powerful method for in-depth proteomics analysis. As part of this strategy, highly efficient and complete proteolytic digestion of proteins into peptides is crucial for successful proteome profiling with deep coverage. To achieve this goal, prolonged digestion time and the use of multiple proteases have been adopted. The long digestion time required and tedious sample treatment steps severely limit the sample processing throughput. Though utilization of immobilized protease greatly reduces the digestion time, highly efficient proteolysis of extremely complex proteomic samples remains a challenging task. Here, we propose a dual matrix-based complementary digestion method using two types of immobilized trypsin with opposite matrix hydrophobicity prepared by attaching trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles. The polymer brushes on the nanoparticles serve as three-dimensional supports for a large amount of trypsin immobilization and lead to ultrafast and highly efficient protein digestion. More importantly, the two types of immobilized trypsin show high complementarity in protein digestion with only ∼60% overlap in peptide identification for yeast and membrane protein of mouse liver. Complementary digestion by applying these two types of immobilized trypsin together leads to obviously enhanced protein and peptide identification. Furthermore, the dual matrix-based complementary digestion shows particular advantage in the digestion of membrane proteins, as twice the number of identified peptides is obtained compared with solution digestion using free proteases, demonstrating its potential as a promising alternative to promote proteomics analysis with higher protein sequence coverage.

A new sample preparation method for the absolute quantitation of a target proteome using 18O labeling combined with multiple reaction monitoring mass spectrometry

“…4 With the development of the proteomic technology, especially the application of ultrahigh-performance liquid chromatography equipped with long chromatographic columns (more than 50 cm in length), packed with sub-2 μm packing materials and capable of ultrahigh resolution, high accuracy and high scan speed mass spectrometry, the digests of protein mixtures can be efficiently separated, identified and quantified in approximately 12 h. [5][6][7] In these cases, the proteolytic steps account for most of the proteomic analysis time, which limits the application of proteomic technologies in real biological samples. 3,8,9 Various materials have been developed as enzyme carriers, including polymer membranes, 10,11 capillary columns, [12][13][14][15][16] microfluidic chips, [17][18][19][20][21] microparticles and nanoparticles, [22][23][24][25][26] porous materials, [27][28][29] graphene oxide [30][31][32][33][34][35][36][37][38][39] and fibers. 3 To address this issue, immobilized enzyme reactors (IMERs) appear to be a better choice for the most efficient and rapid protein digestion at high enzyme to substrate ratios.…”

Achieving efficient digestion faster with Flash Digest: potential alternative to multi-step detergent assisted in-solution digestion in quantitative proteomics experiments

“…Several methods have evolved over the years for increasing the kinetics of proteolytic cleavage, including but not limited to heating, use of detergents, ultrasonication, infrared, microwave, immobilized enzymes and solvent assistance . Immobilized enzyme reactors (IMERs) have been described in great detail as a way to accelerate digestion . Typical systems include an enzyme immobilized (trypsin being widely used, but other enzymes with or without trypsin have been reported) on a suitable stationary phase and a reaction apparatus with a heating element and suitable buffers.…”

“…Typical systems include an enzyme immobilized (trypsin being widely used, but other enzymes with or without trypsin have been reported) on a suitable stationary phase and a reaction apparatus with a heating element and suitable buffers. Enzymes have been immobilized on multiple solid supports including but not limited to suitable polymers, micro/nanoparticles, columns, microchips, porous reactors, beads, graphene and fibers . Immobilization can be achieved through multiple techniques including but not limited to adsorption, encapsulation, covalent bonding, and cross‐linking .…”

“…[2,3,5,6,12,13] Immobilized enzyme reactors (IMERs) have been described in great detail as a way to accelerate digestion. [5,[14][15][16][17][18][19] Typical systems include an enzyme immobilized (trypsin being widely used, but other enzymes with or without trypsin have been reported) on a suitable stationary phase and a reaction apparatus with a heating element and suitable buffers. Enzymes have been immobilized on multiple solid supports including but not limited to suitable polymers, micro/nanoparticles, columns, microchips, porous reactors, beads, graphene and fibers.…”

“…Enzymes have been immobilized on multiple solid supports including but not limited to suitable polymers, micro/nanoparticles, columns, microchips, porous reactors, beads, graphene and fibers. [14][15][16][17][18][19][20][21][22][23] Immobilization can be achieved through multiple techniques including but not limited to adsorption, encapsulation, covalent bonding, and cross-linking. [16,18,19] Immobilization is advantageous because it reduces trypsin-trypsin interactions which in turn reduces autolysis and stabilizes the enzyme, thus maintaining its activity.…”