Innovating the Concept and Practice of Two-Dimensional Gel Electrophoresis in the Analysis of Proteomes at the Proteoform Level

Proteomes. 2019 Oct 30;7(4):36. doi: 10.3390/proteomes7040036.


Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given "protein" is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (Mr ). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and Mr . Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes.

Keywords: SILAC; TMT; bottom-up proteomics; iTRAQ; isoelectric focusing; liquid chromatography; mass spectrometry; post-translational modification; protein speciation; protein species; proteoform; splicing; top-down proteomics; two-dimensional gel electrophoresis.

Publication types

  • Review