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Proteomes. 2019 Oct 30;7(4). pii: E36. doi: 10.3390/proteomes7040036.

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

Author information

1
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China. yjzhan2011@gmail.com.
2
Hunan Engineering Laboratory for Structural Biology and Drug Design, Central South University, 87 Xiangya Road, Changsha 410008, China. yjzhan2011@gmail.com.
3
State Local Joint Engineering Laboratory for Anticancer Drugs, Central South University, 87 Xiangya Road, Changsha 410008, China. yjzhan2011@gmail.com.
4
National Clinical Research Center for Geriatric Disorders, Central South University, 88 Xiangya Road, Changsha 410008, China. yjzhan2011@gmail.com.
5
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China. libiaomo@foxmail.com.
6
Hunan Engineering Laboratory for Structural Biology and Drug Design, Central South University, 87 Xiangya Road, Changsha 410008, China. libiaomo@foxmail.com.
7
State Local Joint Engineering Laboratory for Anticancer Drugs, Central South University, 87 Xiangya Road, Changsha 410008, China. libiaomo@foxmail.com.
8
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Central South University, 87 Xiangya Road, Changsha 410008, China. xiaohanzhan@gmail.com.
9
Hunan Engineering Laboratory for Structural Biology and Drug Design, Central South University, 87 Xiangya Road, Changsha 410008, China. xiaohanzhan@gmail.com.
10
State Local Joint Engineering Laboratory for Anticancer Drugs, Central South University, 87 Xiangya Road, Changsha 410008, China. xiaohanzhan@gmail.com.
11
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. hschluet@uke.de.
12
Max Planck Unit for the Science of Pathogens, 10117 Berlin, Germany. jungblut@mpiib-berlin.mpg.de.
13
Departments of Health Sciences and Biological Sciences, Faculties of Applied Health Sciences and Mathematics & Science, Brock University, St. Catharines, ON L2S 3A1, Canada. jcoorssen@brocku.ca.

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.

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

PMID:
31671630
DOI:
10.3390/proteomes7040036
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