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 (FASP), 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 cut-off) 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 to the membranebased commercial ultracentrifugation cartridge, our microfluidic device offered a better proteome coverage with ten times less starting material and eight times faster protocol duration.
Metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity in extracting proteins from heterogenous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to LC-MS/MS can successfully be used for identification of microbial proteins. Using cultures of E. coli, B. subtilis and S. cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow higher number of proteins and peptides identification than standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amount of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, in particular in the case of low volume or low cell density.
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