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

Abstract

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.

ChipFilter: Microfluidic Based Comprehensive Sample Preparation Methodology for Metaproteomics

“…The design and fabrication methodology of the microfluidic device has been explained previously (Ndiaye et al, 2020). For the comprehensive sample preparation, cells suspended in ABC were directly loaded into the device in a total volume of 30 μl.…”

“…Microfluidic technology has been successfully applied for the separation of bacterial and viral particles from bioaerosols (Hong et al, 2015), physical cell lysis (Grigorov et al, 2021 for a review) and chemical sample processing by utilizing immobilized trypsin (Huang et al, 2006). In a previous work (Ndiaye et al, 2020), we proposed a ChipFilter Proteolysis (CFP) microfluidic device as a reactor for the miniaturization of protein sample processing and digestion steps. The CFP design is closely related to the experimental setup of filter-aided sample processing (FASP).…”

Dual roles of drug or its metabolite−protein conjugate: Cutting‐edge strategy of drug discovery using shotgun proteomics

“…Until now, there have been four methods that can achieve peptide desalination and fractionation in the same device, including spintip-based proteomics technology (SISPROT), 146 iST, 112 S-Trap, 109 and on-chip. 147 For instance, the detection of membrane proteins, including many oncogenic proteins, is increased by reducing the sample loss using SISPROT. 146,148 This easy-to-use technology identified 3771 annotated membrane proteins from 100,000 stem cells.…”

“…Based on online MD‐LC, integrated proteomics sample preparation and fractionation methods are rapidly being developed to reduce sample loss and time and increase enzymatic efficiency. Until now, there have been four methods that can achieve peptide desalination and fractionation in the same device, including spintip‐based proteomics technology (SISPROT), 146 iST, 112 S‐Trap, 109 and on‐chip 147 . For instance, the detection of membrane proteins, including many oncogenic proteins, is increased by reducing the sample loss using SISPROT 146,148 .…”

Tackling Current Biomedical Challenges With Frontier Biofabrication and Organ-On-A-Chip Technologies

“…More recently, Ndiaye et al presented a ChipFilter Proteolysis (CFP) microfluidic platform as a proteomics bioreactor for the miniaturization of protein extraction steps for bottom-up proteomics approaches. This system allows the combination of a molecular filtration membrane in PDMS microchip, using soft lithography and replica molding, promoting efficient protein retention and proteolysis on the membrane, and reducing significantly the time for proteome analysis and the amount of the samples if compared with membrane-based commercial ultracentrifugation cartridges ( Ndiaye et al, 2020 ).…”