Serum and plasma proteins/peptides reflect physiologic or pathologic states in humans and are attractive targets for the discovery of disease biomarkers and bioactive proteins/peptides. However, detailed analyses of proteins/peptides in serum/plasma are still challenging because of the presence of many high-abundance proteins, the large dynamic range of protein/peptide concentrations, rendering the extremely high complexity of the proteome. Therefore, we established pretreatment strategies to analyze low abundance proteins/peptides in serum/plasma and evaluated them using SDS-PAGE. Here, the strategies and their application studies to discover biomarker proteins/peptides in serum/plasma are reviewed.

In eukaryotic cells, a wide array of extracellular stimuli generates intracellular signals that converge on a limited number of protein kinase cascades, commonly referred to as mitogen-activated protein kinase (MAPK) pathways. MAPK cascades (MAPKKK-MAPKK-MAPK) are the major signaling systems that dictate cell fate decisions such as, proliferation, differentiation, and apoptosis. There are at least three subfamilies of MAPKs, named p38, JNK, and ERK in human cells. The ERK subfamily members are activated by mitogenic stimuli and are associated with cell proliferation. In contrast, JNK and p38 are activated by environmental stresses (e.g., DNA damage or osmotic shock) and by cytokines (e.g., TNFα), and play pivotal roles in cellular stress responses. Thus, p38 and JNK are collectively called stress-activated MAPKs. Perturbation of these critical signaling systems is involved in a variety of life-threatening disorders, including cancer and auto-immune diseases. Therefore, these signaling systems are of clinical importance. We previously identified several molecules that regulate MAPK pathways, such as MTK1, GADD45, PP2C, and MCRIP1, and showed that some of these molecules were indeed aberrantly regulated in human cancer. In this review, I will discuss resent findings concerning the regulation of MAPK signaling in cell fate decision and outline some of the major findings from our laboratory.

To separate and analyze high-molecular-weight (HMW) proteins (>200 kDa), we developed an N,N′-methylenebisacrylamide gradient gel electrophoresis with a constant percentage of agarose and acrylamide (BIS-gradient APAGE). We used a 0–0.24% BIS gradient gel with with 0.5% agarose and 4.5% acrylamide to yield a good resolution of proteins more than 200 kDa up to 400 kDa. When the BIS-gradient APAGE system was used for the second dimension of two-dimensional gel electrophoresis with agarose gel in the first dimension (Agarose 2-DE), chicken atrial and ventricular myosin heavy chain isoforms were successfully separated from each other. Moreover, the BIS-gradient APAGE gel is easier to handle than the conventional 5% SDS-PAGE gel. The BIS-gradient APAGE system has a good resolution, low cost, and high reproducibility. Therefore, the use of the BIS-gradient APAGE would be one of the good choice for proteomic analysis of HMW proteins.

Exosomes are extracellular vesicles that contain proteins and nucleic acids and are surrounded by a double-layer membrane of the original cells. Therefore, the exosomes are thought not only as the novel biomarker, but as a gene delivery vehicle that contribute to a cell-to-cell interaction in tumor cells. The purpose of this study is to reveal the contribution of exosomes for systemic complications of multiple myeloma with a focus on sugar chains in exosomes. To determine the appropriate conditions for exosome isolation, we used culture media with or without FBS and isolated exosomes by ultracentrifugation. The amounts of protein in exosome fractions were similar in both conditions, revealing that the sugar chains on exosomes can be analyzed using culture media without FBS which contain miscellaneous proteins including sugar-binding protein. When myeloma cells were stimulated by IL-6 in FBS-free condition, the amount of sialic acid expressing on the cell surface increased depending on time and concentration of IL-6. Whereas the amount of sialic acid in exosome fraction decreased at 2 days but clearly recovered in 4 days. These results suggest that exosomes derived from myeloma cells partially reflect the alteration of sugar chains on the original cells due to cancerous cytokine stimulation, and also suggest the possibility that the exosomes relate to the exacerbation of tumor progression and development of systemic complications.

JOKOH CO., LTD. was established in 1947 as a distributor of X-ray film and medical equipment. In 1964, we started to sell the cellulose acetate membrane, which was first developed by Fujifilm Corporation in Japan. The needs for the automation of the electrophoretic measurement increased as the electrophoresis using the cellulose acetate membrane became popular. For this needs, we first developed and sold the instrument, which automates the measurement of the optical density of stained protein on the membrane. By using this instrument, the time and effort for calculation of percentages in the fraction patterns was dramatically improved. In this post, we introduce the history of our research and development in automation of serum protein electrophoresis on the cellulose acetate membrane.

Cellulose acetate membrane (CAM) electrophoresis for protein fraction is an easy and simple method. We have classified urinary protein patterns into glomerular, tubular, and mixed, using the relative mobility of each band on CAM electrophoresis of urine samples from patients with different types of kidney diseases. To apply this classification method in the future, we analyzed urine samples from individuals with either orthostatic proteinuria or Dent disease. The samples from individuals with orthostatic proteinuria were analyzed using SDS-PAGE and detected specific 27.7 kDa and 39.2 kDa bands. In addition, when urine samples of 15 min and 30 min after lordotic load testing were analyzed using CAM electrophoresis, these protein fractionations showed glomerular injury pattern. CAM electrophoretic analysis of urine samples from patients with Dent disease showed a urinary protein fraction different from serum protein fraction. Slow α₂ and β fractions of this analysis were characterized. Furthermore, in urine from patients with Dent disease, cystatin C and prealbumin were detected using a natural contact blotting method. Prealbumin was detected in the slow α₂ fraction in addition to the prealbumin fraction. Subjecting urine sample of patients suffering from kidney diseases to an exhaustive analysis by CAM electrophoresis possibly is an extremely effective method in kidney disease diagnosis.

Electrophoresis has been less commonly employed in laboratories. Reasons for this include its complicated procedures, high cost, and the advent of other specific tests. However, the use of electrophoresis is convenient and effective to elucidate the causes of abnormal biochemical values. In addition, no subsequent scrutiny is required when the causes of abnormalities can be identified by electrophoresis, reducing patients’ burdens and worries. In this article, to verify the usefulness of electrophoresis in routine laboratory tests, we examined cases for which electrophoresis was successfully employed to elucidate the causes of abnormal biochemical values in our hospital.

Genetic testing has been expanded by the technological advances of gene analysis. The quality of the nucleic acid is important to secure quality of the analysis, and preservation and the transportation of the clinical sample influence the quality of the nucleic acid. In recent years, for the purpose of the quality control of the nucleic acid, on-tip electrophoresis such as Bioanalyzer (Agilent Technologies) is used. Because analysis by the next-generation sequencing is used in clinical inspection, these techniques are necessary for the quality control of analysis data. In other words, the technique of the electrophoresis continues being used in a genetic testing while progressing.

We have developed a fully automated microfluidic immunoassay system, μTASWako i30, by employing disposable plastic microfluidic chips. The system relies on the Liquid-phase Binding Assay - Electrokinetic Analyte Transport Assay (LBA-EATA) principle and can provide assay results with a short turnaround time (TAT) within 9 min. The key assay components of this method are the fluorescent dye-labeled antibody for Laser-Induced Fluorescence (LIF) detection and the DNA-coupled antibody. DNA is a highly negatively charged molecule, therefore, the DNA-coupled antibody and its immunocomplex behave like a DNA molecule in electrophoresis even if protein is attached on it. It contributes to improve sensitivity and separation efficiency in CE immunoassay. DNA-coupled antibody is highly concentrated by Isotachophoresis (ITP), and the immunocomplex is formed when DNA-coupled antibody passes through the sample zone. The immunocomplex is also concentrated by ITP and it is separated from non-specific fluorescence substances by switching CE mode from ITP to gel electrophoresis. In the AFP-L3% assay and PIVKA-II, we could realize short assay time (TAT is 9 min), high sensitivity (at the pM level) and good reproducibility (CV: 0.6–6%). Five assay items, AFP-L3, PIVKA-II (des-gamma-carboxy prothrombin: DCP), PCT (procalcitonin), N-terminal pro-BNP (NT-proBNP) and TroponinT (TnT) are available in the market for μTASWako i30.