Woodland Breyer, Necakov Aleksandar, Coorssen Jens R
Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, ON L2S 3A1, Canada.
Ronin Institute, Montclair, NJ 07043, USA.
Proteomes. 2023 Mar 6;11(1):10. doi: 10.3390/proteomes11010010.
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.
整合自上而下蛋白质组学是一种分析方法，它能充分满足有效且常规评估蛋白质组所需的广度和复杂性。尽管如此，任何此类评估也需要对方法进行严格审查，以确保尽可能深入地进行定量蛋白质组分析。在此，我们为蛋白质组提取物建立了一种优化的通用方案，以改善蛋白质异构体的减少，从而提高二维电泳（2DE）中的分辨率。在将二硫苏糖醇（DTT）、三丁基膦（TBP）和2-羟基乙二硫醚（HED）单独及组合使用之前，先在一维SDS-PAGE（1DE）中进行测试，然后再将其应用于完整的2DE方案中。与文献中报道的其他条件和还原方案相比，在样品复水前，用100 mM DTT + 5 mM TBP进行还原可增加斑点数量、总信号和斑点圆度（即减少拖尾）。数据表明，就蛋白质异构体减少而言，许多广泛应用的还原方案明显“动力不足”，因此限制了常规自上而下蛋白质组分析的质量和深度。
