Robust centrifugal microfluidic miniaturization and automation of target enrichment for protease substrate identification

• Applicants: Professor Dr.-Ing. Nils Paust; Professor Dr. Oliver Schilling
• Subject Area: Microsystems
• Term: from 2018 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 405351425

Proteins constitute the functional and structural foundation of living systems. Proteomics is the system-wide analysis of proteins; typically performed by mass spectrometry (MS) to identify 1000s of proteins in minute sample amounts. Modern mass spectrometers are powerful but sample preparation lacks far behind with regard to robustness and reproducibility. This project aims to study centrifugal microfluidic automation of sample preparation for the MS based analysis of proteolytic processing. Proteolysis is the enzyme-catalyzed cleavage of proteins into smaller fragments. Often, these cleavage products are stable and possess novel functionality. Dysregulated proteolysis is a hallmark of numerous diseases such as cancer. MS-based methods to study proteolysis on a proteome-wide scale have recently been invented. To study native proteolytic processing, protein termini are tagged and enriched in complex workflows. Although these schemes are now an integral part in many research efforts, they remain laborious and suffer from low robustness and poor reproducibility. These drawbacks are a major hurdle in using MS-based proteomics to study disease-associated proteolysis in clinical specimens, for which often only limited sample material is available. Our research project studies centrifugal microfluidic automation of complex protein-chemical workflows for highly precise and robust sample preparation. More than 30 processing steps (chemical protection of terminal peptides, washing, solid phase extraction) adding up to more than 100 unit operations (liquid transport, metering, mixing, valving) are integrated into centrifugal cartridges. Based on our experience in centrifugal microfluidics as well as in proteomics, we anticipate that centrifugal microfluidic automation will significantly enhance consistency of the complete analysis chain while at the same time minimizing sample consumption. We aim to ultimately employ centrifugal microfluidic sample processing to study key steps of proteolytic processing in clear cell renal cell cancer, using precious patient samples.Our research will focus on overall system integration, on downscaling of volumes to the microliter range, and on simultaneously studying the impact of each automation step (such as metering, mixing, affinity based separation) on the biochemical performance of the analysis. Thereby, the enrichment strategy of chemically protecting the target peptides of the protein, digesting the protein into peptides and removing all non-protected fragments (negative selection) is investigated with microfluidic automation for the first time. Eventually, we aim to specifically remove more than 90 % of the peptides in the sample in order to perform a focused analysis of the remaining 10 % (here: protein terminal peptides). We anticipate that the proposed research will have a substantial impact in both areas, in centrifugal microfluidics a well as in proteolysis research.

• DFG Programme: Research Grants