Comparative Analysis of Proteoform Clean-Up Methods for Improved Top-Down Proteomics

Proteoforms, the endogenous forms of polypeptides that take into account all sources of variation (genetic and chemical), may be more relevant for understanding complex biological mechanisms, including disease phenotypes, than generically defined “protein families”. Mass spectrometry-based top-down proteomics often utilizes electrophoretic techniques that rely on sodium dodecyl sulfate (SDS) to fractionate proteoforms prior to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Methanol-chloroform-water (MCW) precipitation is commonly utilized for SDS removal due to its efficacy and low cost, yet it may lead to poor recovery of smaller proteoforms. Here, four commercial SDS clean-up alternatives were benchmarked against MCW. Results suggest that MCW yields fewer proteoform identifications, particularly among small and acidic proteoforms. The analysis of post-translationally modified proteoforms identified using the different clean-up methods indicates increased prevalence of methylation modifications post-MCW clean-up. Among the commercial kits, DetergentOUT and HiPPR achieved SDS removal comparable to MCW but at a higher cost. For studies sensitive to loss of low molecular weight or acidic proteoforms, these kits may offer an advantage. Alternatively, at a lower cost, MinuteSDS still provides sufficient SDS removal and broader proteome coverage.