Smart Trypsin Adsorption into N‐(2‐Aminoethyl)‐3‐aminopropyl‐Modified Mesoporous Silica for Ultra Fast Protein Digestion

Abstract

Comprehension of protein networks and expression patterns in cells, tissues, and organisms is essential for technological applications of modern life sciences. Due to the recent developments over the past few years, mass-spectrometry-based proteomics has expanded its interface role to broad and diverse research areas of science and technology. [1][2][3] Even so, to satisfy the demand for high-speed analysis, new flexible and efficient tools are an urgent priority.Peptide mass fingerprinting remains the most powerful and straightforward method for protein mass identification. Specifically, proteolytic digestion generates characteristic peptide fragments that are typically acquired by matrix-assisted, laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and matched to theoretical digestion patterns in a database.[4] However, conventional protein digestion protocols need exceedingly long digestion times that limit high-throughput protein identification. The use of trypsin immobilized onto solid support has recently captured the attention of many research groups, because these systems can significantly speed-up digestion.[5]Herein, we report for the first time the application of trypsin adsorbed on N-(2-aminoethyl)-3-aminopropyl-derivatized mesoporous silica particles (MPs) used as an exceptional enzymatic bio-reactor for protein digestion. Proteolytic fragments of myoglobin were obtained as quickly as 1 min from the addition of the protein to the trypsin-immobilized mesoporous support, giving a 100 % sequence coverage. Furthermore, the substantial contribution of N-(2-aminoethyl)-3-aminopropyl and aminopropyl (indicated as AAPTES and APTES, respectively) in SBA-15-functionalized materials to proteolysis was evaluated in comparison to nonfunctionalized SBA-15. This reactor system has great potential for significantly increasing proteolytic efficiency compared to similar biocatalysts, as well as providing a flexible and rapid approach to the field of digestion techniques for MS-based proteomics. The main goal of this study was to design a simple and fast approach for protein digestion as a part of an on going project aimed to the development of convenient and sensitive procedures for proteomics applications. [6][7][8] MPs, characterized by a well-ordered porous structure and extremely high surface area, are excellent candidates for biomolecules and enzyme immobilization.[9] While trypsin-immobilization strategies mostly rely on covalently linked trypsin to the support, [5] only very few examples of physical adsorption of trypsin onto a porous support have been reported for MS-based protein analysis. [10,11] The use of covalent bonding between the enzyme and the support, while reducing enzyme leaching from the support, has been reported to modify the conformation of the enzyme with reduction of catalytic activity. [9,12] Moreover recent studies suggest that mesoporous structures with a pore size close to the enzyme dimension provide stable enzyme adsorption with high efficiency. [13][14][15] Sta...

Recent advances of mesoporous materials in sample preparation

“…Following by pH change and applying trypsin, the denaturation and concurrent proteolysis of broad-range proteins could be efficiently achieved, resulting in the identifications of a broad range of diverse proteins with high sequence coverage by mass spectrometry. Similarly, trypsin immobilization on SBA-15 or amine and cyano functionalized SBA-15 materials have also been applied for protein proteolysis [54][55][56]. Casadonte et al [54] found that the amine functionalized SBA-15 showed higher digestion efficiency than unmodified SBA-15 with the same pore diameters.…”

“…Similarly, trypsin immobilization on SBA-15 or amine and cyano functionalized SBA-15 materials have also been applied for protein proteolysis [54][55][56]. Casadonte et al [54] found that the amine functionalized SBA-15 showed higher digestion efficiency than unmodified SBA-15 with the same pore diameters. Also, the proteolytic efficiency of functionalized SBA-15 was 1000 times faster than that of using conventional free solution method.…”

Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion

“…More importantly, some IMERs could be readily coupled to separation and identification systems, enabling fast, efficient, high-throughput and automated proteome analysis [7]. Till now, enzymes have been immobilized on different supports, such as membranes [8], carbon materials [9], monolithic materials [10][11][12][13][14][15][16][17], polymers [18][19][20][21], silica materials [22,23] and hybrids [24]. However, the improved binding capacity of enzymes and the decreased peptide residue on matrices are still the hot-points under serious consideration in this field.…”

Process development for the obtention and use of recombinant glycosidases: expression, modelling and immobilisation

“…The use of aminated organosilicas to adsorb acidic enzymes was recently reported (Casadonte et al, 2010). In this contribution, a simple protocol based on the use of carbodiimides to couple amino groups with the free carboxyl groups of the enzymes enables fast and efficient covalent binding on the NUVM-7 supports.…”

“…In these supports, immobilisation is usually carried out by adsorption based on electrostatic affinity between the amino groups of the enzyme and the hydroxyl groups of the support. The support can be functionalised with amino groups or activated with glutaraldehyde to link them to carboxyl groups of the enzyme (Subramanian et al, 1999;Casadonte et al, 2010).…”