Centrifugal microfluidic automation of the protein aggregation capture workflow for robust mass spectrometry-based proteomics

Abstract

Proteomic sample preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly addressed by automated approaches. However, in clinical settings for precision medicine, where a limited number of samples must be processed in a standardized and reproducible manner with minimal user interaction, fully automized workflows remain scarce. Here, we present the AutoPAC-disk, a centrifugal microfluidic implementation of a protein aggregation capture (PAC) sample preparation workflow for bottom-up proteomics that automates all necessary steps for on-bead proteolysis including on-disk pre-storage of buffers. Comparative evaluation of the AutoPAC-disk using HEK293 cell lysates against a manual reference workflow and a semi-automated robotic PAC implementation showed 50% and 37% more peptide identifications and 23% and 10% more protein group identifications, respectively, while maintaining high quantitative reproducibility as reflected by protein-group intensity coeffincients of variation (CVs) below 10%. Additional analysis demonstrated that the AutoPAC-disk primarily increased identifications of low-abundance proteins without introducing method specific physicochemical bias. The AutoPAC-disk was subsequently evaluated using patient-derived formalin-fixed paraffin-embedded (FFPE) prostate tumor tissue. The AutoPAC-disk yielded 8% more peptide identifications and 10% more protein groups than the manual workflow, with protein-group intensity CVs below 7% for both methods. Together, these results demonstrate that centrifugal microfluidic automation with on-disk buffer pre-storage can substantially simplify proteomic sample preparation, minimize user interaction and lower operational barriers for personnel with limited experience in proteomic sample preparation, providing a promising strategy for clinical and translational proteomics in the field of precision medicine.