Accelerated protein digestion by numbering-up nanochannels, followed by LC–MS analysis

Techniques for analyzing proteins, including single-cell proteomics, are increasingly important and employed in diverse fields. The time-consuming nature of protein digestion presents a bottleneck in proteomics studies, however. To accelerate digestion reactions, reactors using packed beads or monolithic columns with immobilized enzymes are often used, but controlling the reaction remains challenging due to non-uniform gap sizes. We previously developed a picoliter nanofluidic digestion method, but comprehensive analysis of protein digestion using this method was difficult due to limited detection methods in the nanochannels. In this study, we developed a thin-layer nanofluidic device with a nanochannel width of 1.2 mm and depth of 310 nm to increase the product volume to the microliter scale. The nanofluidic device was successfully fabricated via APTES modification, vacuum ultraviolet patterning, washing, bonding, and enzyme immobilization. With the volume-up and numbering-up of the nanochannels, nL/min nanofluidic flow was observed and well controlled over a 16 min reaction. Digested cytochrome c in the nanochannels was collected at microliter scale, which enabled the use of conventional analyses such as liquid chromatography-mass spectrometry. Comparison of the reaction rates for a nanochannel-digested sample and a bulk-digested sample revealed 12-178 times faster reaction in the nanochannels, even when the digestion was performed in nanochannels under acidic conditions. The reason for the accelerated reaction rate remains unclear, but the unique properties of nanochannels may hold the key to elucidating the reaction mechanism.