2DE: the phoenix of proteomics

Abstract

Given the rapid developments in mass spectrometry (MS) in terms of sensitivity, mass accuracy, and throughput, some have suggested that two-dimensional gel electrophoresis (2DE) may no longer be a method of choice for proteomic analyses. However, as recognition of issues with these newer shotgun-MS approaches grows, there is a fresh and growing regard for the maturity of 2DE-MS as a genuine top-down analytical approach, particularly as it resolves thousands of intact protein species in a single run, enabling the simultaneous analysis of total protein complement, including isoforms and post-translational modifications. Given the strengths of both, it is most appropriate to view these as complementary or at least parallel approaches: as proteins encompass a myriad of physico-chemical properties, and the real aim is to explore proteomes as deeply as possible, all available resolving strategies must be considered in terms of the complexity encountered. It is time to critically and constructively focus on the optimization and integration of existing techniques rather than simplistically suggesting that one should replace the other. Our intention here is thus to present an overview of protein resolving techniques, focusing on milestones associated with 2DE, including pros, cons, advances and variations, in particular relative to shotgun proteomic approaches.

Biological significance: Proteomic researchers recognize the importance of 2DE in the history of proteomics. But the latest developments in mass spectrometry-based techniques have led some researchers to retire 2DE in their labs. However, we argue here that 2DE-MS is a genuine top-down analytical approach. The significance of this discussion is to make proteomic researchers aware of the importance of this technique in a proteomic pipeline. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Keywords: 2DE; Bottom-up proteomics; Mass spectrometry; Post-translational modifications; Protein isoforms; Shotgun proteomics; Top-down proteomics; Two-dimensional gel electrophoresis.