Universal solid-phase protein preparation (USP(3)) for bottom-up and top-down proteomics
Dagley, LF; Infusini, G; Larsen, RH; Sandow, JJ; Webb, AI
Journal of Proteome Research
Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP(3)) method which is rapid, robust, and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis. This technique builds upon the single-pot solid-phase-enhanced sample preparation (SP3) where we now demonstrate its scalability (low to high micrograms of protein) and the influence of variables such as bead and enzyme amounts on the efficiency of protein digestion. We also incorporate acid hydrolysis of DNA and RNA during complete proteome extraction resulting in a more reliable method that is simple and easy to implement for routine and high-throughput analysis of proteins. We benchmarked the performance of this technique against filter-aided sample preparation (FASP) using 30 mug of total HeLa protein lysate. We also show that the USP(3) method is compatible with top-down MS where we reproducibly detect over 1800 proteoforms from 50 mug of HeLa protein lysate. The USP(3) protocol allows for efficient and reproducible data to be generated in a cost-effective and robust manner with minimal down time between sample collection and analysis by MS.