On-Chip Sample Preparation Using a ChipFilter Coupled to NanoLC-MS/MS for Bottom-Up Proteomics

Sample
preparation is a crucial step in bottom-up proteomics. Analytical
performances of bottom-up proteomics can be improved by the miniaturization
of sample preparation. Many microfluidic devices have been designed
in the field of proteomics, but many of them are not capable of handling
complex samples and do not integrate the processing and digestion
steps. We propose a ChipFilter Proteolysis (CFP) microfluidic device
as a proteomics reactor for the miniaturization of protein sample
processing and digestion steps, whose design is closely related to
the experimental setup of filter-aided sample processing, even if
no denaturing surfactant is required. The microchip has two reaction
chambers of 0.6 μL volume separated by a protein filtration
membrane in regenerated cellulose (10kD cutoff) that will concentrate
or retain large polypeptides and will release small molecules. Cell
lysis, protein concentration, and rapid chemical or enzymatic treatment
can be performed in the ChipFilter. Complex proteomic samples like
yeast protein extract or whole human cells proteome have been successfully
analyzed with our microchip. Compared with the membrane-based commercial
ultracentrifugation cartridge, our microfluidic device offered a better
proteome coverage with 10 times less starting material and 8 times
faster protocol duration.