This review describes the application of electrophoresis in a clinical laboratory to detect microheterogeneity of protein or sugar chain structure. We developed convenient method of isoelectric focusing using cellulose acetate membrane combined with various staining methods and improved transfer method with PVDF membrane. These methods enabled the easy analysis of clinical samples, and we have explored new biomarkers for various diseases. Target biomolecules of isoelectric focusing was as follows: sugar chains of α₁-acid glycoprotein and antichymotrypsin for inflammation, urinary transferrin for renal diseases, and sugar chains on M protein for multiple myeloma (MM).

We revealed that sugar chains with highly reactive to Con A lectin was detected in the early stage of inflammation, while in the recovery phase of inflammation, sugar chains with highly reactive to DSA lectin increased. For isoelectric point of urinary transferrin, we found that transferrin with low isoelectric point increased in the early stage of nephropathy. Further, when glycosylation of M protein in the samples of MM patients with osteolysis was examined, sugar chains in the light chain on M protein showed high reactivity with Con A lectin. Thus, the analysis of protein binding sugar chains using isoelectric focusing is applicable to develop new biomarkers of various diseases, such as inflammation, nephropathy and cancer.

Protein kinase expression and activity play pivotal roles in various cellular events thorough regulation of phosphorylation signaling. Aberrant expression and activity of protein kinases cause various diseases such as cancer. Therefore, analysis of protein kinase expression and activity is important for understanding their pathogenesis of these condition. In previous reports, various methods for analyzing protein kinases were developed and used to investigate the expression or activity of many protein kinases. However, a method for analyzing the expression and activation of total cellular protein kinases has not been established. To analyze intracellular protein kinases, we generated monoclonal antibodies, designated Multi-PK antibodies, which recognize a wide variety of protein kinases. These Multi-PK antibodies can be used to profile the protein kinases expressed in cells, identify kinases of special interest, and analyze protein kinase expression and phosphorylation state. Additionally, these antibodies could be useful for analysis of various diseases and cellular functions such as glucotoxicity in type 2 diabetes, pathogenesis of ulcerative colitis and epigenetics. Here we introduce newly analytical methods using Multi-PK antibodies combined with Phos-tag 2D-PAGE to analyze the expression and phosphorylation state of total cellular protein kinases. In this review, we focus on recently developed technologies for kinomics studies that use Multi-PK antibodies, which represent a useful analytical tool.

To gain novel sero-diagnostic markers for lung cancer, membranous proteins from three different histological types of lung cancer cell line were analyzed by the shot-gun proteomics method. Biotinylated membranous proteins in each lung cancer cell lines were collected and identified by the shot-gun analysis. From identified 92 membranous proteins, we focused on CD109 and CD155 proteins. Serum levels of these proteins from patients with lung cancer and healthy controls were also examined by the reverse-phase protein array (RPPA) method. Serum levels of CD109 and CD155 were significantly higher in patients with adenocarcinoma and squamous cell carcinoma of the lung than those in healthy controls. And the area under the curve of receiver-operating characteristic analysis of above tumors to healthy control of CD109 were 0.94 and 0.90, and CD155 were 0.96 and 0.77, respectively. The approach for membranous proteins using the shot-gun proteomics method is useful for acquiring novel sero-diagnostic markers.

Antibody drugs are biological polymer and these are inherently heterogeneity due to the complicated physicochemical modifications such as post-translational modification and higher order structure transformation by disulfide bonds during biosynthesis. In recent years, analytical techniques and evaluation methods are highly demanded in order to analyze the structure of antibody drugs in detail to improve the functionalities of antibody pharmaceuticals, manufacturing processes, and quality control. In this study, we aimed to establish a highly accurate evaluation method of antibody drugs using 2DE technology. The analysis chip which can be used in the 2D electrophoretic automation system (Auto2D) has been developed and its validity was examined.

Capillary isoelectric focusing efficiently analyze heterogeneity of protein drugs. My colleagues and I recently developed a method to couple immunoaffinity capture and separation by isoelectric focusing in a single capillary. The method allows sample pretreatment, i.e., purification and concentration, in a single capillary, in which subsequent separation by isoelectric focusing is achieved. The method adds a new option for the analysis of protein drugs in their production processes and for pharmacokinetics in drug-administered patients, with high precision and a minimum amount of samples using an automated instrument.

Here we introduce in-capillary lectin-binding and exoglycosidase-digestion methods using a plug-plug kinetic mode capillary electrophoresis for the analysis of glycoprotein-derived oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). The application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Reactions with neuraminidase and α/β-galactosidase digestions were completed by separation voltage, but hexosaminidase and fucosidase/mannosidase require more drastic conditions such as zero potential amplification and low mixing voltage application, respectively. The methods were applied to the detection of high-mannose type oligosaccharides in a mixture of glycans and NeuGc and α-Gal epitopes in IgG pharmaceuticals.

Lectin microarray is a glycan profiling technology, which allows rapid and high-sensitive analysis of complex features of glycans without liberation of glycans from proteins. Target samples include various biological products including cell therapy products and antibody drugs. In this review, the application of lectin microarray for glycan analysis of cell therapy products and antibody drugs is described.

Mass spectrometry is one of the most frequently used techniques to characterize several quality attributes related to the structure and heterogeneities of biological products. For example, peptide mapping by liquid chromatography/mass spectrometry (LC/MS) is a commonly used strategy for characterizing the primary structure, disulfide bonds, and post-translational modifications (PTMs), including glycosylation, oxidation, and deamidation. Intact mass analysis is also used to confirm molecular heterogeneities originating from PTMs, such as glycosylations, and intended modifications. In addition, a combination method of the hydrogen–deuterium exchange reaction and peptide mapping by LC/MS (HDX/MS) is currently used to evaluate higher-order structures. Therefore, mass spectrometric methods have become increasingly important for determining structural quality attributes of desired biological products. Such mass spectrometric methods used for characterizing biological products are described in this paper.