Sample preparation and cleanup methods for clinical top-down proteomics.

Introduction: The investigation of different proteoforms in clinical samples is a promising approach to elucidate the molecular mechanisms of diseases. Furthermore, proteoform analysis holds great potential for identifying disease-specific biomarkers and targets for personalized medicine. Despite advances in top-down proteomics (TDP) instrumentation, sample preparation and cleanup remain challenging. Work in this area has focused on developing rapid, cost-effective, and less-labor-intensive protocols aimed at minimizing the introduction of artefactual modifications to endogenous proteoforms or bias in proteoform recovery during sample processing.

Area Covered: To inform the selection of sample processing approaches in clinical TDP, this review summarizes state-of-the-art targeted (i.e. affinity and non-affinity-based enrichment) and untargeted (i.e. gel-based fractionation) sample preparation protocols. In addition, currently available offline and online sample cleanup procedures (e.g. dialysis, solid-phase extraction, filter-aided sample preparation, precipitation, and solid-phase protein preparation) are reviewed, highlighting their effectiveness for desalting and/or detergent removal.

Expert Opinion: TDP demonstrates great potential in the clinical setting due to its ability to capture disease-specific proteoforms commonly overlooked in traditional diagnostic assays. The establishment of standardized guidelines for reproducible clinical TDP workflows is essential to leverage advances in sample preparation techniques and analytical instrumentation to facilitate wider adoption of TDP for clinical applications.