Trypsin Immobilization on Hairy Polymer Chains Hybrid Magnetic Nanoparticles for Ultra Fast, Highly Efficient Proteome Digestion, Facile ¹⁸O Labeling and Absolute Protein Quantification

Abstract

In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification.

Improvement of the stability and activity of immobilized trypsin on modified Fe3O4 magnetic nanoparticles for hydrolysis of bovine serum albumin and its application in the bovine milk

“…2(B). The weight loss for MNPs was about 3.57% of total weight, which was attributed to the loss of possible water residue (Qin et al, 2012). When the temperature rose from 40°C to 900°C, 5.6% weight loss occurred for MNPs-GA due to the partial breakdown of the intermolecular bonds with the successive releasing of volatile fractions.…”

Protein Analysis by Shotgun/Bottom-up Proteomics

“…Digital microfluidics allowed for automation of sample clean-up and protein digestion steps for MALDI to improve sensitivity. 89–93 Covalent and dynamic immobilization of trypsin within microreactors, 94–95 on microparticles 96 and nanoparticles 97 and the use of focused highly-intensity ultrasound 98–99 and microwave 100–103 heating have improved the kinetics of tryptic digestion, reducing digestion time. The immobilization of proteases on microwave-absorbing microspheres and nanoparticles further improved speed and efficiency.…”

Construction of a high-performance magnetic enzyme nanosystem for rapid tryptic digestion

“…Normally, the chemical linkage based strategy was used to graft trypsin molecules, and the magnetic particles were first modified with silane reagents, followed by the grafting of enzyme molecules under strict conditions28. However, the whole preparation process focuses on monolayer modification of the particles with small molecules, which would limit the immobilization amount and reduce the accessibility of protein substrates to the immobilized enzyme2429. Although recent works attempted to increase the amount of the binding enzyme by coating a layer of functional polymer, complex, rigorous and longtime modification procedures were required30, which might change the conformation of the enzymes and cause a reduction of their catalytic activity.…”