Towards an optimized workflow for middle-down proteomics

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, either targeting relatively small peptides (i.e. 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e. 10 kDa < Mw < 30 kDa), respectively termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e. 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similiar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates we explored the use of the proteases Asp-N and Glu-C, and a non-enzymatic acid induced cleavage. To increase the depth of the proteome, an SCX separation, carefully tuned to improve the separation of longer peptides, combined with RP-LC using columns packed with material possessing a larger pore size were used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques HCD, ETD and EThcD for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, amongst other things, the enhanced analysis of (co-occurring) post-translational modifications