Phosphorylation is an important post-translational modification of proteins involved in a wide variety of biological processes. The phosphorylation status reflects the relative activities of protein kinases and phosphatases, resulting in the diversity of proteins in the cell. Changes in phosphorylation status may also affect protein function by changing its conformational state, stability, or interactions with other molecules. Various analytical strategies have been proposed to analyze the phosphorylation status of proteins. Here, we will introduce powerful analytical tools for phosphoproteomics based on electrophoresis and mass spectrometry to obtain information on the protein phosphorylation involved in biological processes. Together, we believe that these analytical methods have the potential to accelerate the progress of proteomics research.

By developing the equipment and constructing sample-specific pretreatment methods, we have developed a two-dimensional electrophoresis (2DE) method for the social implementation with (1) high-throughput, (2) high sensitivity, and (3) high reproducibility performance. Furthermore, by taking advantage of the fact that the data obtained by 2DE are images that are compatible with machine learning and the high-throughput performance of the previously developed method, we have succeeded in AI-aided proteomic disease diagnosis using 2DE images of sepsis patient sera. In machine learning, the accuracy and quality (the types of things that can be discriminated) improve as the amount of data increases, and since the 2DE system can in principle be miniaturized, and the practical application of the system can foster big data, it is expected that proteomics will be implemented in society using general-purpose 2DE in the near future. This paper introduces the results of the authors’ research and development to date and discusses new developments in 2DE driven by open innovation accelerated by AI.

The Laminin (Lm)-γ2 chain is associated with Lm-α3 and Lm-β3 chains to form Lm-332, an extracellular matrix protein. Lm-332 has the function of maintaining the structure of biological tissues. On the other hand, Lm-γ2 chain is expressed as a single chain (Lm-γ2m) in invasive cancer and promotes malignant progression of tumor cells by activating the epidermal growth factor receptor (EGFR) signals. Furthermore, we found that the LAMC2 gene encoding the Lm-γ2 chain is expressed specifically in ovarian cancer cells as a fusion gene, LAMC2-NR6A1, which is generated by a chromosome translocation. There is a particularly high level of Lm-γ2m in serum in patients with hepatocellular carcinoma (HCC) compared to healthy donors, and the evaluation can predict future incidence and distant extrahepatic metastases of HCC. This indicates that Lm-γ2m can make a significant contribution to improving the diagnosis and prognosis of HCC as a new serum biomarker. In this review, we will introduce the molecular mechanisms by which the Lm-γ2 chain and the LAMC2-NR6A1 fusion gene product regulate tumor malignant progression, and clinical usefulness of HCC diagnosis as serum biomarker.

PCR-electrophoresis analysis is an effective tool in the education of genetics and genome literacy, since variants of human genomic DNA can be visually observed as the profiles of the bands. It is a useful tool for the teaching of genetics and genome literacy in school. In the United States, variant analysis experimental kits and inexpensive, compact PCR devices are commercially available and used in schools. In the National Science Teachers Association (NSTA), electrophoresis often features in teacher training sessions for Science, Technology, Engineering, and Mathematics (STEM) education relating to understanding the human genome. The analysis of the experimenter’s own genomic DNA is effective in fostering literacy around genetics and the human genome. In Japan, experiments that handle genomes for educational purposes should also be carried out in accordance with official guidelines regarding dealing with personal genetic information, and appropriate methods of operation should be discussed and confirmed. In the future, it will be increasingly necessary to deepen our understanding of electrophoresis, experimental methods, the handling of personal genetic information, and the training of instructors in educational settings.

The separation of native proteins in cytosol of mouse liver using microscale non-denaturing two-dimensional electrophoresis (2DE) requires optimization according to the charges and molecular sizes of the target proteins by adjusting the mixture of ampholytes and the density of acrylamide. Isoelectric focusing (IEF) separation of sorbitol dehydrogenase, lactate dehydrogenase and malate dehydrogenase was accomplished when 5.0% pharmalyte pH 3–10 and 1.3% pharmalyte pH 5–8 were mixed. Additionally, the malate dehydrogenase and carboxylesterase were separated using the 2DE by successively examining their activities after the size-separation of proteins using a 6.6% acrylamide gel. Furthermore, a complex of transferrin and carboxylesterase was separated at the same position on the 2DE after a mixture of transferrin and cytosol proteins in mouse liver separated by the 2DE was detected by reversible staining with imidazole-zinc following carboxylesterase activity staining. This 2DE separation is a first step in the analysis of protein-protein interactions.

The techniques of electrophoresis have been used extensively in the agricultural sciences, from basic research through to practical applications. Agarose gel electrophoresis has been used in studies of plant breeding, including genetically modified crops, and acrylamide gel electrophoresis has been applied to the analysis of proteins in crops and foods. However, the area covered in agricultural sciences have been very wide, with materials and subjects having been set to target many crops and several livestock. This review has introduced electrophoretic applications used in agricultural sciences that have not been used in other areas of life sciences. Six topics were taken up in this review, which were the electrophoresis in soil to remove heavy metals, the native electrophoresis for the analysis of nitrous oxide production affected to global warming, soil microflora, agarose gel electrophoresis for the mapping of quantitative trait loci in crops, mass spectrometry for the analysis of silk, and capillary electrophoresis-mass spectrometry for food analysis.

Tests for measuring immunoglobulins include quantitative immunoglobulin assays, serum protein fractionation tests, immunoelectrophoresis, and immunofixation electrophoresis. The presence of abnormal immunoglobulins is suspected when abnormal bands are detected in electrophoresis or when discrepant data appear between tests. Abnormal immunoglobulins include heavy chain disease proteins and half-molecule immunoglobulins that show molecular defects. Such abnormal proteins can be molecularly qualified by SDS-PAGE or immunoblotting. Bence Jones proteins with microheterogenicity can also be demonstrated as the migration changes by immunofixation electrophoresis of samples before and after enzyme treatment. Furthermore, after agarose membrane electrophoresis, the protein can be spontaneously transferred to a polyvinylidene difluoride (PVDF) membrane for easy immunoblotting. In other words, electrophoresis can be used for both detection and analysis of abnormal immunoglobulins.