The resurgence of two-dimensional gel electrophoresis: Understanding proteins at the proteoform level

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Two-dimensional gel electrophoresis (2DE) is a useful and well-established method for the separation of proteins in a sample, resulting in a 2D protein “spot map”. Recently, Professor Xianquan Zhan of Central South University and Shandong First Medical University, China, found that 2DE allows far more insight into proteins than was previously thought. In fact, each “spot” may contain several hundred proteoforms, or different forms of a protein, rather than just one or two. This discovery has widespread implications across the biomedical sciences.

Since the mid-20th century, gel electrophoresis has been an important tool for researchers hoping to learn more about DNA and proteins. The basic idea behind the method is that large molecules – such as DNA, RNA and proteins – can be separated according to their charge and size. This is achieved by placing the molecules onto a gel and then applying an electric field to the gel. The molecules are separated according to their isoelectric point and move towards the positively charged side of the gel – smaller molecules move quickly while larger molecules move more slowly – to be effectively separated according to their size.

Students of biology are often introduced to gel electrophoresis in one of its more high-profile applications: DNA fingerprinting. This refers to the process of analysing DNA found at crime scenes. However, gel electrophoresis has many other important uses, in a range of different research areas. In recent years, scientists who study the vast and varied group of molecules called proteins have discovered new ways to apply gel electrophoresis technology to their research.

Fig 1. Each 2DE spot contained over 50 to several hundreds of proteoforms. Coomassie blue stained 2-DE pattern of a glioblastoma proteome analysed with IPGstrip pH 3-10 NL and 12% gel concentration of SDS-PAGE. Spots L1-L5 came from one gel for MS/MS analysis. Each spot labelled with a red or green number was combined from three matched gel spots for MS/MS analysis. Modified from Zhan X, et al., Electrophoresis, 2018; and Zhan, et al., Med One, 2018.

Revealing the complexity of proteins
One key step forward was the development of two-dimensional gel electrophoresis (2DE). In this well-established method, a mixture of proteins can be separated by two properties and in two dimensions. Simply put, this allows proteins to be separated at a greater level of detail than would be possible with ordinary gel electrophoresis. The result of the process is a two-dimensional “protein map” in which each protein is represented as an individual “spot”. This technique can be used, for example, to compare the proteins present in a person or animal with a particular disease, such as cancer, to those present in the “normal” condition.

“The proteome is now known to be even more complex than it might
at first appear.”

Professor Xianquan Zhan of Central South University and Shandong First Medical University, China, is an expert in cancer proteomics: the identification and analysis of the proteins associated with cancer, at all stages of the disease. Over a number of years, Prof Zhan has led important research into the use of 2DE in the in-depth study of proteins.

The 2DE method can be used to study the proteome: the entire set of proteins expressed by (i.e. produced by) an organism. However, the proteome is now known to be even more complex than it might at first appear. Proteins can, in fact, be further described according to their proteoforms: all the different molecular forms in which a protein produced by a particular gene might appear. These forms all have slightly different properties, which means that they can be effectively separated by 2DE. In recent years, Prof Zhan has targeted his research towards the study of human proteoforms.

Fig 2. Growth hormone proteoforms derived from the growth hormone gene are found in 24 different 2DE spots.
Modified from Zhan X, et al., Proteomics, 2005.

Understanding proteoforms
In 2005, Prof Zhan and colleagues used 2DE to investigate the various different forms of human growth hormone (hormones are one type of protein). As a result, the researchers were able to successfully identify 24 different human growth hormone proteoforms. This work, which furthered the understanding of the role of human growth hormone in various diseases, laid the foundation for the research that was to follow.

Later, Prof Zhan led an investigation into the proteins associated with a type of often-fatal brain cancer called an astrocytoma. The goal was to identify particular proteins that could be indicators, or potential targets for new treatments, for the disease. The team used 2DE to isolate and identify the important proteins with encouraging results. At the same time, the researchers gained a crucial insight: they discovered that each 2D spot on the protein map contained from 50 up to several hundred unique proteoforms rather than simply one or two different proteins as was previously thought. This insight demonstrated that 2DE could describe the complexity of proteins in much greater resolution: at the level of the proteoform.

Fig 3. 2DE has the huge potential for the large-scale study of proteoforms.
Modified from Zhan et al., Proteomes, 2019.

To confirm this exciting discovery, Prof Zhan and colleagues next carried out a series of experiments using 2DE in combination with mass spectrometry, a powerful method that can both quantify and identify different compounds within a sample. This rigorous testing confirmed that each 2DE spot can contain hundreds of different proteoforms (see figure 1). Further, the results showed that the majority of these proteoforms are low, or extremely low, in abundance – and therefore may not be picked up by traditional methods.

The team also realised that proteoforms derived from the same gene could be found in different 2DE spots (see figure 2). In fact, each 2DE spot could contain different proteoforms created from the same gene, proteoforms from different genes, or both.

The revival of 2D gel electrophoresis
In the last few decades, while 2DE remained a popular and reliable method, in some fields of research it was superseded by newer techniques. This was because 2DE was widely considered to be a “low throughput” option, as it typically allowed for the identification of just one or two proteins per spot. Researchers in the field of proteomics (the large-scale study of proteins) in particular turned to other, “high throughput” methods that were seemingly better suited to the vast diversity of proteins.

“When combined with the newest, high-specificity forms of mass spectrometry, 2DE can be used to analyse the breadth of the human proteome at the proteoform level.”

The work of Prof Zhan and his colleagues has changed this. Thanks to their discovery that several hundred proteins or proteoforms could be identified in a single 2DE spot, 2DE has proven to be well equipped to handle the demands of modern protein research. When combined with the newest, high-specificity forms of mass spectrometry, 2DE can be used to comprehensively analyse the breadth of the human proteome at the proteoform level. In 2018, Prof Zhan and his fellow researchers were able to set out their argument for the revival of two-dimensional gel electrophoresis in the large-scale study of proteoforms (see figure 3).

Fig 4. Concept and formation of proteoforms. PTM: Post-translational modifications.
Modified from Zhan et al, Med One, 2018; and Zhan et al., Proteomes, 2019.

From Protein to Proteoform
It is now clear that any one protein actually represents a “family” of proteoforms (see figure 4). This means, according to Prof Zhan, that the proteoform, rather than the protein, should be viewed as the base unit of the proteome. This idea, which represents a shift in thinking in the field of proteomics, is supported by the fact that proteoforms have different properties (and can therefore be separated by 2DE).

Prof Zhan suggests that 2DE should be viewed as the initial – and crucial – step in the separation of proteins. Cutting-edge mass spectrometry can then be used to identify each proteoform. Further, Prof Zhan proposes that 2DE and mass spectrometry should be used in conjunction with a technique called stable isotope labelling. This method involves labelling molecules such as proteins with isotopes (particular variations of a chemical element) in order to detect differences in the abundance of proteins between samples. Together, these techniques would form a powerful toolkit with which to separate, identify and quantify proteoforms, allowing the most in-depth analysis of the proteome that is currently possible.

The idea of the proteoform is relevant across the biomedical sciences. For researchers like Prof Zhan, who study widespread and serious diseases such as cancer, a better understanding of the role of proteoforms in disease is crucial to future progress. Thanks to the revival and renewed potential of 2DE, this improved understanding is within reach. In future, Prof Zhan hopes to continue to delve further into the complex and varied world of proteoforms.


Are there any specific ways in which the study of proteoforms has led to new treatments, for example for cancer?

Yes. The study of proteoforms will directly lead to the discovery of reliable biomarkers for accurate understanding of molecular mechanisms, the discovery of effective therapeutic targets, and for effective prediction, diagnosis, and prognostic assessment. For example, the man-made therapeutic proteins, including insulin, monoclonal antibodies, erythropoietin, and human growth hormone, have a huge number of proteoforms within a single recombinant therapeutic protein. The in-depth study of proteoforms of therapeutic proteins will directly improve their therapeutic efficiency in treatment of disease such as cancer. To give another example, the study of human hormone proteoforms (such as growth hormone – GH, and prolactin – PRL) will directly benefit the deep insight into hormone-related disease such as GH or PRL-secreting pituitary adenomas. Also, the serum hormone proteoform pattern (such as GH, or PRL) has an obvious potential to develop as biomarkers for prediction, diagnosis, and prognostic assessment of GH or PRL-related diseases.

 

References

  • Zhan, X., Giorgianni, F., Desiderio, D.M. (2005). Proteomics analysis of growth hormone isoforms in the human pituitary. Proteomics, 5, 1228–1241. Available at: https://doi.org/10.1002/pmic.200400987
  • Qian, S. et al. (2018). Prolactin variants in human pituitaries and pituitary adenomas identified with two-dimensional gel electrophoresis and mass spectrometry. Frontiers in Endocrinology, 9, 468. Available at: https://doi.org/10.3389/fendo.2018.00468
  • Zhan, X., Long, Y., Lu, M. (2018). Exploration of variations in proteome and metabolome for predictive diagnostics and personalised treatment algorithms: Innovative approach and examples for potential clinical application. Journal of Proteomics, 188, 30–40. Available at: https://doi.org/10.1016/j.jprot.2017.08.020
  • Peng, F. et al. (2015). Nitroproteins in human astrocytomas discovered by gel electrophoresis and tandem mass spectrometry. J. Am. Soc. Mass Spectrom., 26(12), 2062–2076. Available at: https://doi.org/10.1007/s13361-015-1270-3
  • Zhan, X. et al. (2018). How many proteins can be identified in a 2-DE gel spot within an analysis of a complex human cancer tissue proteome? Electrophoresis, 39, 965–980. Available at: https://doi.org/10.1002/elps.201700330
  • Zhan, X., Li, N., Zhan, X., Qian, S. (2018). Revival of 2DE-LC/MS in proteomics and its potential for large-scale study of human proteoforms. Med One, 3, e180008. Available at: https://doi.org/10.20900/mo.20180008
  • Zhan, X. et al. (2019). Innovating the concept and practice of two-dimensional gel electrophoresis in the analysis of proteomes at the proteoform level. Proteomes, 7(4), 36. Available at: https://doi.org/10.3390/proteomes7040036
  • Zhan, X. (ed.). (2020). Proteoforms: Concept and Applications in Medical Sciences. InTech – Open science publisher, London, United Kingdom. ISBN: 978-1-83880-034-5. Available at: https://doi.org/10.5772/intechopen.83687

Research Objectives

Prof Zhan is introducing a new concept of two-dimensional gel electrophoresis (2DE) for proteomics to decipher proteoforms.

Funding

  • Hunan Provincial Hundred Talent Plan
  • Xiangya Hospital Funds for Talent Introduction
  • China “863” Plan Project (Grant No. 2014AA020610-1)
  • National Natural Science Foundation of China (Grant No. 81272798 and 81572278)
  • Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008)
  • Shandong First Medical University Talent Introduction Funds

Collaborators

  • Prof Dominic M. Desiderio from University of Tennessee Health Science Center, USA
  • Prof Olga Golubnitschaja from University of Bonn, Germany
  • Prof Peter R. Jungblut from Max Planck Unit for the Science of Pathogens, Germany
  • Prof Hartmut Schlüter from University Medical Center Hamburg-Eppendorf, Germany
  • Prof Jens R. Coorssen from Brock University, Canada

Bio

Xianquan Zhan received his MD and PhD training in preventive medicine at West China University of Medical Sciences during 1989-1999. He received his post-doctoral training in oncology and cancer proteomics at Central South University and University of Tennessee Health Science Center (UTHSC). He worked at UTHSC and Cleveland Clinic in USA during 2001-2012 and achieved the rank of Associate Professor at UTHSC. In 2012, he moved to Central South University and Shandong First Medical University as a Professor and Advisors of MS/PhD graduate students and postdoctoral fellows. He is also the Fellow of Royal Society of Medicine, Fellow of EPMA, European EPMA National Representative, Full member of ASCO, AAAS member, Editor-In-Chief of IJCDT, Associate Editors of EPMA Journal and BMC Medical Genomics, and Guest Editor of Frontiers in Endocrinology and Mass Spectrometry Reviews. He has published 135 articles, 23 book chapters, and 2 US patents in the field of clinical proteomics and biomarkers, with more than 2382 citations, and H-index 28.

Prof Zhan’s main research interest focuses on the studies of cancerproteomics and proteoforms, multiomics and biomarkers, and the use of modern omics techniques and systemsbiology for predictive, preventive and personalised medicine (PPPM) and precision medicine (PM) in cancer.

Prof Xianquan Zhan

Contact
Xianquan Zhan
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health
Central South University
87 Xiangya Road
Changsha 410008
P.R. China

University Creative Research Initiatives Center
Shandong First Medical University
6699 Qingdao Road
Jinan, Shandong 250117
P.R. China

E: yjzhan2011@gmail.com
T: +86 15675860818
W: https://www.researchgate.net/profile/Xianquan_Zhan2

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