Chararacterization of a monolithic immobilized trypsin microreactor with on‐line coupling to ESI‐MS

Abstract

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 lm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one-or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto-to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 258C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 378C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.

Enzymatic microreactors in biocatalysis: history, features, and future perspectives

“…Bound by the APTES/GA method to micro and nano-magnetic particles [127] Coupled to the polymer by reaction with GA [125,157], or by using commercial TPCK-trypsin [100]. Poly(methyl methacrylate) Encapsulated in silica gel anchored to the polymer surface as such [99], or modified by zeolite [102].…”

“…In silica microreactors, the most frequently used method requires activation by (3-aminopropyl) triethoxysilane (APTES) and glutaraldehyde (GA), but analogous reactions with imides or epoxides have also been reported [94][95][96][97]. Similar methods were also used to functionalize polycarbonate with a carbodiimide [98] and poly(methyl methacrylate) with several copolymers [99][100][101].…”

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.…”

Silica-Immobilized Enzyme Reactors

“…The resulting immobilized enzyme reactors (IMERs) can also be integrated directly to further analytical methods such as liquid chromatography or mass spectrometry. [6] In fact, one of the primary applications of many IMER systems is the digestion of proteins for automated sequence analysis by integration with mass spectrometry. The use of IMERs for protein digestion, however, has been reviewed extensively elsewhere and will not be covered herein.…”

“…The use of IMERs for protein digestion, however, has been reviewed extensively elsewhere and will not be covered herein. [6][7][8][9][10] A variety of matrices have been investigated over the years for enzyme immobilization or encapsulation, but for focus, the emphasis within this review is limited to IMERs that utilize silica as a scaffold. The use of silica-based IMERs has received increasing attention since their appearance in the early 1970's and since then, the use and diversity of applications has grown steadily ( Figure 1).…”