An aptamer-based trypsin reactor for on-line protein digestion with electrospray ionization tandem mass spectrometry

An immobilized trypsin reactor that was based on aptamers has been developed and applied for the first time for proteomic digestion. Briefly, 25 single-stranded DNA aptamers that were specific for trypsin were obtained after SELEX (systematic evolution of ligands by exponential enrichment) selection. TApt.23 (no. 23 trypsin aptamer), which had the lowest dissociation constant (Kd) value (0.0123 μM), was amino-modified and subsequently grafted to an amino-modified silica surface with glutaraldehyde. The results indicated that 14.65 ± 0.35 μg of trypsin could be immobilized on 10 mg of TApt.23-silica when an optimized borate buffer was used. Subsequently, a trypsin reactor was fabricated by using a PEEKsil micro column. Compared with in-solution digestion, the aptamer-based trypsin reactor exhibited similar results for protein identification but used a much shorter digestion time (∼30 min). An on-line analysis platform, which included a trypsin reactor coupled to a high-performance liquid chromatography tandem mass spectrometry device, was built through a 6-port valve and achieved efficient protein digestion compared with in-solution and off-line methods. Compared with irreversible covalent enzyme immobilization, the aptamer-based carrier enables more rapid and convenient immobilized trypsin elution as well as re-immobilization of the enzyme. This superior reactor demonstrated that an aptamer could become a more widely used method for enzyme immobilization and other applications.