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

A significant and well-established technological platform allowing comprehensive top-down proteomic analysis is the two-dimensional gel electrophoresis (2DE). However, 2DE’s long-held but now increasingly obsolete traditional principles have clearly impacted its application at the level of protein species/proteoforms to in-depth investigations of proteomes. It is time for a new definition of 2DE for proteomics to be popularized. Any provided “protein” is now recognized to consist of a series of proteoforms with the production and enrichment of the proteome concept. Thus, it is the proteoform that is the fundamental unit of a proteome, rather than the canonical protein, and each proteoform has a particular isoelectric point (pI) and relative mass (Mr). Each proteoform can therefore be systematically resolved and arrayed according to its distinct pI using 2DE and Mr. Each observable spot contains several proteoforms derived from the same gene as well as from various genes. In a 2DE pattern, proteoforms derived from the same gene are distributed into various spots. High-resolution 2DE is thus essentially an initial degree of separation to resolve proteome complexity and is basically a form of pre-fractionation prior to analysis using mass spectrometry (MS). In addition, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has enormous potential for the proteoforms that constitute proteomes to be detected, classified, and quantified on a large scale. Overall, the intrinsic capacity to draw on the synthesis of refined top-down and bottom-up analytical methods is the beauty and strength of this coupled 2DE-LC-MS/MS technique. The capacity at the vital proteoform level for genuine deep proteome analysis is now a definite fact.

Author (s) Details

Xianquan Zhan
Science and Technology Innovation Center, Shandong First Medical University, 6699 Qingdao Road, Jinan 250117, China and Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China and State Local Joint Engineering Laboratory for Anticancer Drugs, Central South University, 87 Xiangya Road, Changsha 410008, China and National Clinical Research Center for Geriatric Disorders, Central South University, 88 Xiangya Road, Changsha 410008, China.

Biao Li
Science and Technology Innovation Center, Shandong First Medical University, 6699 Qingdao Road, Jinan 250117, China and Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China and State Local Joint Engineering Laboratory for Anticancer Drugs, Central South University, 87 Xiangya Road, Changsha 410008, China.

Xiaohan Zhan
Science and Technology Innovation Center, Shandong First Medical University, 6699 Qingdao Road, Jinan 250117, China and Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China and State Local Joint Engineering Laboratory for Anticancer Drugs, Central South University, 87 Xiangya Road, Changsha 410008, China.

Hartmut Schl├╝ter
Institute of Clinical Chemistry and Laboratory Medicine, Mass Spectrometric Proteomics, Campus Forschung, N27 Raum 00.008, University Medical Center Hamburg-Eppendorf, Martinistr, 52, 20246 Hamburg, Germany.

Peter R. Jungblut
Max Planck Unit for the Science of Pathogens, 10117 Berlin, Germany.

Jens R. Coorssen
Departments of Health Sciences and Biological Sciences, Faculties of Applied Health Sciences and Mathematics & Science, Brock University, St. Catharines, ON L2S 3A1, Canada.

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