Autoantibodies (AAbs) are antibodies detected in sera from patients with various autoimmune diseases. Because they are also observed in sera from patients with various malignancies before cancer-associated antigens can be detected and found even in sera up to five years before symptomatic disease, many researchers have paid attention to serum AAbs for early cancer detection. In this review, we described our methodology to detect early tumor-specific, chemo-sensitivity predictive, and prognostic autoantigens that recognized by autoantibodies in sera from patients with lung and bladder cancers using by two-dimensional gel electrophoresis combined with immunoblot analysis.

Abnormalities in protein post-translational modifications (PTMs) are both causes and consequences of disease. Therefore, it is essential to clarify how PTMs influence protein functions by comprehensively collecting information concerning them. Mass spectrometry (MS) allows us to obtain a great deal of information regarding the PTM sites on various proteins. However, results of PTM analysis have not been utilized efficiently to understand the functional regulation of proteins. To address this deficiency, we developed the ModProt (Post-Translational Modification Map of Proteome) database, which integrates our laboratory data and literature information about PTM sites, and subsequently constructed a web-based laboratory information management system (LIMS) that allows us to administer the ModProt database and view PTM site maps. Furthermore, several differences in PTM status under different conditions, such as healthy and diseased states, can be detected by comparing PTM data from this database. The ModProt database helps us to manage our own laboratory information obtained through proteome-wide PTM analysis, and to obtain further insights into specific PTM sites that may be important to protein functions. We believe that the ModProt database will serve as a powerful tool for basic biomedical research and advanced clinical investigations based on protein PTM data in the context of personalized medicine.

MEK1, a chief component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cell lines. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites (phosphoproteomic data), permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. The Phos-tag-based strategy, which effectively uses phosphoproteomic data, permits qualitative and quantitative time-course profiling of intact MEK1 phosphospecies molecules in their respective phosphorylation states without loss of information about the molecular masses of the protein species. This combined study involving a Phos-tag-gel-based approach and existing phosphoproteomic data previously obtained by MS-based phosphoproteomic methods has shed new light on the phosphorylation dynamics of MEK1.

Biomarkers are essential tools to achieve correct diagnosis, and the development of cancer biomarkers has been one of the most major topics in cancer prtoteomics. Many efforts and research budgets were devoted to biomarker studies in the last decade. However, there is no biomarker, which was developed by proteomics and proven to be clinically useful. The limitations of modalities and validation have been considered factors to hinder the successful biomarker study. However, the fundamental problem of biomarker studies in the laboratory was not well considered. In the most of biomarker studies, almost equal number of cases and controls are compared to identify the biomarker candidates. When high sensitivity and specificity are obtained by the biomarkers created in that condition, those biomarkers are considered to be candidates to be promising. However, the number of cancer patients in the ordinary population is extremely lower than non-cancerous persons, and the sensitivity and specificity calculated in the laboratory conditions may not make sense in the clinical setting. We need to employ the research theme in which the clinical situation can be reproduced in the laboratory. Such theme may include the biomarkers for differential diagnosis, and prediction of response to treatments or prognosis. Proper stratification of samples based on the statistical thought also improves the study for early detection. In conclusions, to obtain the clinically useful biomarkers by cancer proteomics, we need to consider the realistic utility of biomarkers in the clinical setting.

To develop sero-diagnostic markers for lung cancer, surface proteins were labeled and collected, then identified by the shotgun analysis. Surface proteins were biotinylated from three each of lung cancer cell lines and collected them. Biotinylated proteins were identified by the shotgun analysis. Expression levels of identified proteins in four typical lung cancer cell lines were confirmed by immunoblotting and immunohistochemistry. And also examined CD109 levels as a sero-diagnostic marker for lung cancer, sera from 268 lung cancer patients and 100 healthy controls were analyzed by the reverse-phase protein array (RPPA) method. Serum levels of CD109 were significantly higher in patients with adenocarcinoma and squamous cell carcinoma than in healthy controls (p < 0.0001), and the area under the curve (AUC) of receiver-operating characteristic curve (ROC) analysis was 0.93, when an optimal cut-off value of 1.13 was applied, the diagnostic sensitivity and specificity for lung cancer were 84 and 88%, respectively. In addition, because CD109 was detected even in stage I disease, serum levels of CD109 antigen should be applicable early sero-diagnostic markers for lung cancer patients discriminating from healthy controls. To our knowledge, this is the first report providing evidence that CD109 may be an early sero diagnostic marker for lung cancer.

Various cellular events are regulated through protein phosphorylation mediated by protein kinases. These protein kinases are known to be involved in the pathogenesis of many diseases. Although 518 protein kinase genes were identified in the human genome, it remains unclear how many and what kind of protein kinases are expressed and activated in cells under varying situations. To investigate cellular phosphorylation signaling, we established three hybridoma cell lines (M1C, M8C, YK34) producing monoclonal antibodies, designated Multi-PK antibodies that can recognize multiple protein kinases in diverse biological species. Specifically, the M1C and M8C antibodies recognize Ser/Thr kinases and the YK34 antibody detects Tyr kinases. Using Multi-PK antibodies, newly analytical methods were developed: a profiling method for analysis of protein kinase expression in cells; a method for protein kinase identification by 2D-PAGE in combination with cyanogen bromide digestion of protein kinases; and an analytical method for intracellular protein kinase expression and phosphorylation state. In addition, we introduce three applications of Multi-PK antibodies to identify and characterize the protein kinases involved in epigenetics, glucotoxicity in type 2 diabetes, and pathogenesis of ulcerative colitis. In this review, we focus on the recently developed technologies for kinomics studies using the powerful analytical tools of Multi-PK antibodies.

Cow’s milk allergy is one of the most common food allergies found in children. Children with a history of anaphylactic shock to cow’s milks having a reaction to milk-specific IgE are reported to be at high risk of severe and persistent cow’s milk allergy. Cow’s milk is a highly nutritious food; however, it is also one of the most common food allergens. The major allergens from cow’s milk have been found to be β-lactoglobulin and caseins. In this paper, we have examined how bromelain can reduce the major allergen in milk.

The existence of the cancer stem cell relates to prognosis and resistance to chemotherapy and radiotherapy. However, the existing cancer stem cell is very few in a cancer tissue, and it is difficult to conduct various studies. In this study, we aimed at the acquisition of the candidate marker protein to detect a cancer stem cell, we established cancer stem cell-like micropapillary adenocarcinoma cell line using reprogramming factor-induced iPS cells. Here, we compared protein profiles with cancer stem cell-like micropapillary adenocarcinoma cell line and the parent cells to discover biomarker proteins.