Immobilization of Trypsin on Superparamagnetic Nanoparticles for Rapid and Effective Proteolysis

Abstract

In this work, a novel and facile route was developed for the immobilization of enzyme on nanosized magnetic particles, and its application to fast protein digestion via a direct MALDI-TOF mass spectrometry analysis was demonstrated. At first, amine-functionalized magnetic particles with high magnetic responsivity and excellent dispersibility were prepared through a facile one-pot strategy. Then, magnetic nanoparticles were functionalized with numerous aldehyde(-CHO) groups by treating the as-synthesized, amine-functionalized magnetic nanoparticles with glutaraldehyde. Finally, immobilization of trypsin onto the aldehyde-functionalized magnetic nanoparticles was achieved through reaction of the aldehyde groups with amine groups of trypsin. The obtained trypsin-immobilized magnetic nanoparticles were conveniently applied for protein digestion. The digestion efficiency was demonstrated with peptide mapping analysis of three model proteins. The process of digestion is very facile due to the easy manipulation of magnetic nanoparticles. Complete protein digestion was achieved in a short time (5 min), without any complicated reduction and alkylation procedures. These results are expected to open up a new possibility for the proteolysis analysis as well as a new application of magnetic nanoparticles. Additionally, it is worth noting that, since the preparation and surface functionality of magnetic nanoparticles is low-cost and reproducible, the preparation method and application approach of the magnetic nanoparticles may find much potential in proteome research.

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

“…MALDI-TOF MS analysis also showed high sequence coverage (89%). The value of sequence coverage was higher than the other trypsin-immobilized reactors reported (Li et al, 2007a;Liu et al, 2009) and the same as that performed by in-solution digestion (37 ºC for 18 hours), suggesting that the immobilized TY showed rapid and efficient proteolysis. The multi-digestion of Cyt-C by the tandem microreactor that was connected by using TYmicroreactor and CT-microreactor was evaluated.…”

“…Similar to the digestion of lysozyme, the digestion of BSA by TY-microreactor at 50 °C efficiently occurred but not at 30 °C. The sequence coverage of 37% (201/583 amino acids) was higher compared with that of in-solution digestion (26%, 151/583 amino acids) and was better or comparable to those of a thermal (Liu et al, 2009;Sim et al, 2006) or chemical denatured BSA (Li et al, 2007a;Chatterjee et al, 2010) or the reduced BSA (Ma et al, 2008) by the reported trypsin-microreactors (24-46%). In addition, the number of identified disulfide bonds was 10 of 17, which is superior to 6 obtained by in-solution digestion.…”

“…These results suggest that the stability of immobilized proteases is superior to that of free proteases. A typical sample preparation for proteolysis before digestion involves multi-steps including denaturation, reduction of disulfide bond, and alkylation of free thiol group to reduce the conformational stability of protein; steps which are expected to produce enhancement of digestion efficiency (Ma et al, 2008;Li et al, 2007a;Ethier et al, 2006;Lin et al, 2008). However, the multi-step procedure is time-consuming.…”

“…Several methods for protease immobilization have been reported, wherein the protease, usually trypsin, has been immobilized in microchips by sol-gel encapsulation (Sakai-Kato et al, 2003;Wu et al, 2004), covalently bounded (Lee et al, 2008;Fan & Chen, 2007) and physically adsorbed onto different supports . In addition, trypsinimmobilized magnetic particles have been developted to carry out proteolysis with a short digestion time (Li et al, 2007a;. However, preparations of these protease-immobilized microreactors require multi-step procedures consuming considerable amounts of time and effort.…”

A novel organic-inorganic hybrid monolith for trypsin immobilization

“…In addition, the IMERs can be reused hundreds of times. To date, many IMERs [4][5][6][7][8][9][10][11][12] have been designed and applied successfully. Monolith based IMERs [8][9][10][11][12] prepared in capillaries have been particularly popular because of the monolithic relatively high binding capacity for enzymes, low backpressure, biological inertia, and mechanical stability.…”