Clinical peptidomic analysis by a one-step direct transfer technology: its potential utility for monitoring of pathophysiological status in female reproductive system disorders.

To date, numerous studies have searched for candidate molecules or clinical examination methods as potential biomarkers for monitoring intractable diseases, such as carcinomas. Evidence accumulated over the past decade shows that many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. Although an analysis of the whole peptide (the 'peptidome') using mass spectrometry is thought to be one of the most powerful and promising experimental approaches, it has failed to identify biomarkers in the clinical blood samples, presumably due to the methodological limitations. In general, commonly used techniques for proteomic analysis of blood require the removal of large amounts of serum/plasma proteins prior to mass spectrometry analysis, and this step seems to have resulted in the overlooking of important biomarkers during the analytical process. Here, we provide a brief overview of a new quantitative peptidomic analysis by a one-step direct transfer technology without depletion of major blood proteins. Using this technology, we herein report experimental data on serum peptidomic analysis for patients with pregnancy-induced hypertension as a clinical model. In addition, we refer to the potential utility of this approach for the monitoring of pathophysiological status in female reproductive system disorders in general.

Overexpression of TEX101, a potential novel cancer marker, in head and neck squamous cell carcinoma.

TEX101, a member of the Ly-6/urokinase-type plasminogen activator receptor (LU)-family we previously identified, is a germ cell-marker glycoprotein. To date, it is reported that some members of the protein-family are overexpressed in a variety of cancer tissues. We previously reported Ly6k, a member of the LU-family, as an association molecule with TEX101 in murine male germ cells. LY6K (a human homologue of Ly6k) is overexpressed in head and neck squamous cell carcinomas (HNSCC). These facts led us to speculate that TEX101 may also exist in HNSCC, like LY6K. Using an anti-human TEX101 polyclonal antibody (pAb) established, we examined the expression of TEX101 protein in cancer tissues by immunohistochemical analysis. TEX101 was detected in the cancer cells of some tissue specimens from patients with HNSCC, whereas the normal squamous epithelium was immunonegative. The TEX101 protein was detected in cancer cells from 54 out of 64 (80.6%) patients with HNSCC. The rate of lymph nodes metastasis tends to be low in TEX101-positive patients, compared to patients with weakly positive and negative expression of TEX101. The present results imply that TEX101 is a novel cancer-related protein and may be useful as a marker for prognosis/diagnosis of HNSCC.

Molecular characterization and expression of dipeptidase 3, a testis-specific membrane-bound dipeptidase: complex formation with TEX101, a germ-cell-specific antigen in the mouse testis.

We previously established an anti-sperm head auto-monoclonal antibody designated Ts4. The immunoreactivity of this antibody was also observed in other reproduction-related cells, such as testicular germ cells and early embryos, suggesting that the Ts4-recognized molecules might play a role in the reproductive process. However, the molecular characteristics and functions of the antigens warrant further clarification. In this study, we primarily attempted identification of the mAb-recognized molecules within the mouse testis. An immunoprecipitation method, together with liquid chromatography-tandem mass spectrometry, revealed that the testicular immunoprecipitants with Ts4 contained dipeptidase 3 (DPEP3), a member of the membrane-bound dipeptidase family. A Western blot analysis using an anti-DPEP3 polyclonal antibody established in this study showed that this molecule was glycosylated and formed a disulfide-linked homodimer within the testis. Expression of DPEP3 protein was observed in the testicular germ cells, but not in the Sertoli or interstitial cells, or in any other major organs. Although Western blot analysis of testicular proteins separated by two-dimensional SDS-PAGE failed to demonstrate binding of Ts4 to DPEP3, we found that DPEP3 forms complexes with Ts4-immunoreactive molecules, such as TEX101, on the surfaces of spermatocytes, spermatids, and testicular spermatozoa. Based on data showing in the present study, further studies concerning DPEP3 on the testicular germ cells may help to clarify the molecular mechanisms of testicular germ-cell development.

Quantitative peptidomic analysis by a newly developed one-step direct transfer technology without depletion of major blood proteins: its potential utility for monitoring of pathophysiological status in pregnancy-induced hypertension.

We have recently developed a new target plate (BLOTCHIP®) for MALDI-MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy-induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC-MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen-1 (three peptides), fibrinogen-α, complement component C4-A/B, α-2-HS-glycoprotein and inter-α-trypsin inhibitor heavy chain H4. 2-D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP® analysis can be applied for discovery study of PIH biomarker candidates.

Molecular expression of Ly6k, a putative glycosylphosphatidyl-inositol-anchored membrane protein on the mouse testicular germ cells.

Ly6k, a putative mouse glycosylphosphatidyl-inositol (GPI)-anchored membrane protein is specifically associated with a unique germ-cell marker, TEX101. Although a human orthologue LY6K has been proposed as a novel cancer/testis antigen, its molecular nature is largely obscure, because its characteristics have been gleaned mainly from qualitative studies of gene structure. The aim of this study is to characterize molecular nature of Ly6k more precisely, especially, to focus on the molecular expression during testicular development. The present study have shown that: (1) Ly6k was strongly observed in testis, but faint expression was broadly noticed in other tissues; (2) Ly6k was weakly detected in testes from 18-day postcoitus to 1-day postpartum (dpp), with a plateau starting around 8-dpp; and (3) testicular Ly6k showed two-peak expression at around 14-dpp and 24-dpp, then exhibited stable expression from 6-week after birth onward. Western blot and immunohistochemical analyses revealed that Ly6k exists in at least two forms: a GPI-anchored form (17kDa) and a water-soluble (non-membrane) form (12kDa), and the 17-kDa mature form is expressed in the testicular germ cells beginning approximately 10days after birth. This information is essential for the molecular classification of Ly6k, and may help uncover the detailed physiological role of Ly6k in gametogenesis, or cancer biology.

Distribution of molecular epitope for Ts4, an anti-sperm auto-monoclonal antibody in the fertilization process.

To investigate molecular effects of anti-sperm autoantibodies on fertilization, we previously established anti-mouse sperm-head auto-monoclonal antibodies (mAbs). Among the mAbs established, one mAb (named Ts4) recognized the sugar moiety of TEX101, a germ cell-marker glycoprotein. In the present study, we examined the immunoreactivity of Ts4 in mouse spermatozoa and fertilized eggs during early embryogenesis to clarify the distribution of the Ts4-reactive antigen in the fertilization process. Similar to TES101 mAb (a specific probe for TEX101), immunopositive staining of Ts4 was observed on spermatocytes, spermatids and spermatozoa within the testis. In contrast to the results obtained with TES101 mAb, Ts4 reacted with the sperm acrosomal region within the cauda epididymis. A Western blot analysis of epididymal sperm extract revealed that Ts4 mainly detected two bands between 100 and 150 kDa, while Ts4 faintly detected a band corresponding to TEX101 at 38 kDa. In addition, Ts4-reactive molecules were observed in the growing early embryo after fertilization. Since Ts4-reactive antigen, potentially a carbohydrate chain, is only observed in reproduction-related areas such as the testis, epididymal sperm-head and early embryo, it is expected to have an effect on fertilization. Therefore, additional studies of this antigen may elucidate the molecular mechanisms underlying the reproductive process.