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

Abstract

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 However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms.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.

A database for inventory of proteoform profiles: “2DE‐pattern”

“…In our view, this inventory could use the benefits of our methodology [27]. It should be noted that recent publications of other laboratories have confirmed fidelity of our approach [29]. As we generated a body of information about proteoforms using top‐down proteomics (2DE separation followed by shotgun ESI–LC–MS/MS) that was conveniently organized in a graphical way, we decided to build a database, where that information could be kept and used by the scientific community.…”

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

“…Despite this lack of resolving power when compared to ideality, the separation afforded by 2DGE is clearly an advantage when trying to bring proteomics closer to the proteoform world [ 5 , 6 , 7 ]. Consequently, there are literally thousands of scientific articles that bring together the keywords “2D electrophoresis” and “post-translational modifications”, and this article will of course not aim at citing all of them.…”

“…A decade after its introduction in the mid-1970s [ 1 , 2 ], two-dimensional electrophoresis was instrumental in the birth of proteomics [ 3 ] Since then, the exponential development of shotgun proteomics, pioneered at the turn of the century [ 4 ] and driven in great part by the gigantic increase in the sensitivity of mass spectrometers, has largely superseded two-dimensional (2D) gel-based proteomics. However, although often being referred to as an outdated method, 2D gel-based proteomics still has some merit, as recently reviewed [ 5 , 6 , 7 ]. The purpose of this review is to expand on this topic and emphasize the positive features that make 2D gel-based proteomics an option to consider in some circumstances.…”

Editorial: Molecular Network Study of Pituitary Adenomas

“…However, proteome complexity is significantly influenced by RNA splicing, PTMs, and many other factors (14). The concept development of proteoform/protein species significantly enriches the content of proteome; a protein is an umbrella term of proteoform encoded by the same gene, and a proteoform is defined as its amino acid sequence + PTMs + spatial conformation + cofactors + binding partners + localization + a function, and thus proteoform is the basic unit of proteome (27,28). Clarification of proteoforms and proteoform-mediated signaling pathway networks will precisely help understand the molecular mechanism, directly identify reliable biomarkers for precise diagnosis and prognostic assessment, and precisely determine therapeutic treatment of pituitary adenoma (28,29).…”