Immobilized enzyme reactors for “omics” approaches

During the last decade, miniaturization, availability of new analytical tools as well as introduction of high-resolution instrumentation on the market have made feasible an important step forward in the “omics” field. However, notwithstanding the great advances, experimental workflows still suffer from several problems, which requires improved analytical strategies to be solved. In particular, most protocols in shotgun proteomics and glycomics commonly involve an enzyme-catalyzed key step, which bears several drawbacks such as long incubation times, loss of (expensive) material due to the non-reusability of the enzyme, poor reproducibility and low automation. To circumvent these problems, significant efforts have been recently made to develop more efficient procedures, limit interferences and enhance automation and throughput. Among these, developing immobilized enzyme reactors (IMERs) is an attractive approach. IMERs have shown several advantages over the traditional in-solution methods, among which high enzyme-to-substrate ratio and high efficiency, which translate into shorter analysis time, long term stability, higher reproducibility and higher automation. Thus IMERs, if correctly integrated into a LC-MS analytical platform, may lead to improved workflows in “omics” protocols that involve enzyme-catalyzed steps. In particular, conventional workflows in proteomics include in-gel or in-solution protein digestion by proteases, most commonly by trypsin, while in glycomics Peptide-N-glycosidase F (PNGase F) is commonly used to release glycans from glycoproteins or glycopeptides. IMERs based on such enzymes may, therefore, find interesting applications in proteomics and glycomics. To achieve automation and a significant decrease in analysis time IMERs were prepared using short bed, high performance monolithic columns (CIMac™ Analytical columns), which are suitable supports for IMER preparation when integration into a separative system is required and large molecules need to be analyzed. Different chemistries and immobilization protocols were used to achieve stable IMERs with high enzyme activity. Operational conditions to preserve IMER activity and achieve hyphenation with MS system were optimized. In particular, trypsin-IMERs were obtained by covalent immobilization on CIMac™ analytical columns. Trypsin loading and surface chemistry were shown to be key factors for rapid and efficient protein digestion. Comparison with in solution digestion and a commercially available trypsin-column showed a high efficiency for the new developed IMER, with advantages over conventional methods. On the other hand, the PNGase F-IMER was obtained by oriented covalent immobilization of the target enzyme onto an epoxy CIMac™ analytical column. The PNGase F-IMER could be inserted into a LC-ESI-Q-ToF system, enabling the on-line deglycosylation of target proteins and analysis of the released glycans without any derivatisation step.