Characterization of Reaction Dynamics in a Trypsin-Modified Capillary Microreactor

Abstract

Application of mild vibration to an immobilized trypsin capillary microreactor can enhance digestion rates for many globular and glycosylated proteins (12-70-kDa range) without additional sample handling. A sinusoid wave form generator and a simple piezoelectric transducer were used to apply vibration in a wide frequency range to the 50-μm-i.d. enzyme microreactor over its entire length. The mass transport properties of the microreactor were quantitatively examined for protein digestions through the use of an artificial globular protein. This was synthesized by covering the surface of 35-nm-diameter latex beads with a peptide (Leu-Arg-Leu). Capillary electrophoresis analysis of the microreactor products showed there were no mass transport-related effects for vibration of the capillary. Digestions of a range of globular protein structures were performed and the products analyzed by capillary electrophoresis. The rate enhancements were found to be related to the stability of the protein tertiary and secondary structure. Cytochrome c showed a dramatic acceleration in rate of digestion as the vibration frequency increased over a range of 200 Hz to 7.1 kHz. The ability to enhance reaction rates for very stable proteins can be gained by additional means of destabilizing the protein, as shown by removal of calcium from α-lactalbumin. Vibration of the enzyme capillary will have the greatest utility for extremely limited protein samples since chemical modification to completely denature proteins usually requires considerable sample handling.

Functionalized magnetic micro‐ and nanoparticles: Optimization and application to μ‐chip tryptic digestion

“…A solution-phase enzymatic assay was described by Wang [1], but this approach can yield autodigestion, and may require a delicate separation between the enzyme and the reaction products. The sorption or covalent coupling of enzyme molecules to the inner surface of capillary (microchannel) is possible [35], but is limited by the low specific inner surface of the capillary and the impossibility to renew the enzyme activity. This limitation was overcome by the immobilization of enzyme molecules to the surface of nano/microparticles [33,36].…”

Immobilized metal‐ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization‐time of flight‐mass spectrometry

“…But the bioreactive probes are critical to reuse because of the loss of the enzymatic activity due to the rigorous washing tip process or the occupation of the active site of the proteases by the excessive matrix. Most reported microreactors are based on the format of microcolumns, that is, the enzyme is immobilized on support media [6,7], and then packed into the microcolumn, or onto the surface of silicon-based microfluidic chips [8,9] or on the fused-silica capillary directly by the covalent bonding method [10][11][12]. Compared with digestion of protein by the immobilized enzyme on probe tips, less analyte is needed for the mass mapping and the enzyme is reusable.…”

“…Licklider et al [10] reported the use of a capillary microreactor for protein digestion, followed by online ESI and off-line MALDI analysis. The microreactor is vibrated with an electric engraving tool to enhance reaction rates and reduce the digestion time to about 30 min [11]. Similarly, a thermal denaturation procedure can effectively accelerate the digestion of a protein by a capillary microreactor; by this method 9 fmol horse heart cytochrome c could be well characterized within 5 min [12].…”

Immobilized microfluidic enzymatic reactors

“…Kuhr's group [31,32] later found that the proteolysis reaction rate could be enhanced by applying low-power acoustic vibration to the capillary, with digestion carried out in a batch-wise procedure. Efficient tryptic digestions of large proteins have been carried out in as few as 30 min [33]. The capillary microreactors were used for protein characterization by trypsin, pepsin and carboxypeptidase Y digestion.…”