Optimized Proteome Reduction for Integrative Top-Down Proteomics

Integrative top-down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly 'under-powered' in terms of proteoform reduction and thus, limit the quality and depth of routine top-down proteomic analyses.

Automated summary

Integrative top-down proteomics is an effective approach for analyzing proteomes, and it requires a rigorous review of methodology to ensure accurate quantitative analysis. In this study, an optimized protocol using DTT, TBP, and HED as reduction agents was developed to improve protein resolution in 2DE. Compared to other reduction protocols, this optimized protocol showed improved spot counts, total signal, and spot circularity, indicating its potential for enhancing routine top-down proteomic analyses.