Analytical methods that do not routinely analyze the full breadth of protein species (i.e. proteoforms), beyond identifying canonical amino acid sequences, are unable to analyse the full of proteome. Integrative top-down proteomics using two-dimensional gel electrophoresis (2DE) coupled to liquid chromatography, and tandem mass spectrometry (LC-MS/MS), is currently the only technique that fully addresses the depth needed for effective and routine assessments of the whole proteome. This is due to its well-established capacity to resolve and identify thousands of intact proteoforms. However, a key element in any proteomic workflow is the reduction and alkylation of disulfide bridges. If not completely reduced and alkylated, disulfide bridges can reform, both intra- or intermolecularly creating ‘scrambled bridges’ that can result in additional, non-native species. Thus, an appropriate concentration and combination of effective reducing agent(s) is essential.
In this study, we improved detection of proteoforms by 2DE by optimizing the concentration and combination of reducing agents. To establish a protocol that would be broadly applicable, we tested both mammalian and plant extracts for improved proteoform detection. To determine the treatment yielding optimal resolution, samples were treated with various combinations and concentrations of excess free thiols (dithiothreitol, DTT), trivalent phosphorus reagents (tributylphosphine, TBP) and organic disulfides (hydroxyethyl disulfide, HED).
We were able to determine that reducing proteome extracts with 100 mM DTT + 5 mM TBP yielded increased spot counts, greater total signal, and better spot circularity. Next steps will include comparisons of mass spectrometric data using different reducing agent combinations to demonstrate the efficacy of this technique in identifying proteoforms. These data will then indicate that optimizing the concentration and combination of reducing reagents significantly improves integrative 2DE-based proteome analyses by enabling the routine focus on proteoforms rather than only canonical amino acid sequences.