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.

Hinrichs, M., Wallner, J., Thiery, J., Kleber, C., Metzger, M., Sieber, S.A., Hutzenlaub, T., Schilling, O., Paust, N., Hahne, H., Muntel , J. and  Klatt,  J.-N. “Centrifugal microfluidic automation of the protein aggregation capture workflow for robust mass spectrometry-based proteomics”, Lab Chip (2026).

Link: https://doi.org/10.1039/D6LC00211K

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