Two-dimensional gel electrophoresis in proteomics: past, present and future

Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.

💡 Research Summary

Two‑dimensional gel electrophoresis (2‑DE) has played a pivotal role in the emergence and evolution of proteomics, yet it is no longer the sole separation technology employed in the field. This review provides a comprehensive historical perspective, beginning with the early days when 2‑DE was used before the term “proteomics” even existed. The authors trace the sequence of events that led to the birth of proteomics, highlighting how the combination of isoelectric focusing (IEF) and SDS‑PAGE in the 1970s enabled the simultaneous separation of proteins by isoelectric point and molecular weight. This breakthrough created the first large‑scale protein maps, establishing a protein‑centric view that complemented transcriptomic and genomic studies.

The review then discusses the technological advances that transformed 2‑DE from a labor‑intensive technique into a high‑throughput platform. Automation of gel casting, the development of robust image‑analysis software, and the introduction of sensitive staining methods (Coomassie, silver, and fluorescence dyes) dramatically increased dynamic range and detection limits, allowing low‑abundance proteins to be visualized. Digital imaging and quantitative spot analysis further enabled comparative studies across multiple conditions.

Despite these improvements, the authors acknowledge the well‑known limitations of 2‑DE. Proteins that are extremely hydrophobic, have very high or very low pI values (≤ 3 or ≥ 10), are larger than ~150 kDa, or are expressed at very low levels often escape detection. Gel‑to‑gel reproducibility remains a challenge, and the lack of standardized data formats hampers large‑scale meta‑analyses. Compared with modern LC‑MS/MS workflows, 2‑DE offers lower throughput, limited automation, and a higher demand for manual expertise.

Nevertheless, 2‑DE retains several unique advantages that keep it relevant. First, it separates intact proteins, preserving post‑translational modifications (PTMs) in their native context, which is valuable for PTM‑focused studies. Second, the physical gel serves as a “map” that can be re‑examined, excised, and subjected to downstream mass‑spectrometric identification without additional sample preparation steps. Third, the cost per analysis is considerably lower than that of high‑resolution mass spectrometers, making 2‑DE accessible to laboratories with limited budgets.

The authors propose scenarios where 2‑DE should be the method of choice. When the research goal is to obtain a global overview of protein expression changes, to detect PTM patterns using modification‑specific antibodies, or to perform rapid screening of drug‑induced proteome shifts, 2‑DE offers a combination of robustness, resolution, and ease of interpretation that is hard to match with purely LC‑MS‑based approaches. They also discuss emerging hybrid technologies, such as coupling microfluidic devices with gel electrophoresis to improve automation and reduce sample consumption, and integrating artificial‑intelligence‑driven image analysis to enhance spot detection and quantification.

Looking forward, the review emphasizes the need for community‑wide data sharing initiatives. Public repositories of gel images, coupled with standardized metadata schemas, would enable cross‑laboratory comparisons and meta‑analyses, thereby extending the utility of 2‑DE beyond individual projects. The authors envision that, with continued integration of AI‑based image processing, robotic gel excision, and improved PTM‑specific staining, many of the current reproducibility and throughput constraints can be mitigated.

In conclusion, 2‑DE was the cornerstone of early proteomics, and while it has been eclipsed by high‑resolution mass spectrometry for many applications, it remains a valuable complementary tool. Its ability to separate intact proteins, maintain PTM information, and provide a visual, cost‑effective snapshot of the proteome ensures that 2‑DE will continue to have a niche in precision proteomics, systems biology, and exploratory studies, especially when combined with modern computational and microfluidic enhancements.