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Background: Dialysis-related amyloidosis (DRA) is a severe complication in end-stage kidney disease (ESKD) patients undergoing long-term dialysis treatment, characterized by the deposition of β 2 -microglobulin-related amyloids (Aβ2M amyloid). To inhibit DRA progression, hexadecyl-immobilized cellulose bead (HICB) columns are employed to adsorb circulating β 2 -microglobulin (β2M). However, it is possible that the HICB also adsorbs other molecules involved in amyloidogenesis., Methods: We enrolled 14 ESKD patients using HICB columns for DRA treatment; proteins were extracted from HICBs following treatment and identified using liquid chromatography-linked mass spectrometry. We measured the removal rate of these proteins and examined the effect of those molecules on Aβ2M amyloid fibril formation in vitro ., Results: We identified 200 proteins adsorbed by HICBs. Of these, 21 were also detected in the amyloid deposits in the carpal tunnels of patients with DRA. After passing through the HICB column and hemodialyzer, the serum levels of proteins such as β2M, lysozyme, angiogenin, complement factor D and matrix Gla protein were reduced. These proteins acted in the Aβ2M amyloid fibril formation., Conclusions: HICBs adsorbed diverse proteins in ESKD patients with DRA, including those detected in amyloid lesions. Direct hemoperfusion utilizing HICBs may play a role in acting Aβ2M amyloidogenesis by reducing the amyloid-related proteins.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR., (© 2023. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)

The Human Proteome Project (HPP) has made great efforts to clarify the existing evidence of human proteins since 2012. However, according to the recent release of neXtProt (2019-1), approximately 10% of all human genes still have inadequate or no experimental evidence of their translation at the protein level. They were categorized as missing proteins (PE2-PE4). To further the goal of HPP, we developed a two-step bioinformatic strategy addressing the utilization of the SRMAtlas synthetic peptides corresponding to the missing proteins as an exclusive reference in order to explore their natural counterparts within GPM. In the first step, we searched the GPM for the non-nested SRMAtlas peptides corresponding to the missing proteins, taking under consideration only those detected via ≥2 non-nested unitypic/proteotypic peptides "Stranded peptides" with length ≥9 amino acids in the same proteomic study. As a result, 51 missing proteins were newly detected in 35 different proteomic studies. In the second step, we validated these newly detected missing proteins based on matching the spectra of their synthetic and natural peptides in SRMAtlas and GPM, respectively. The results showed that 23 of the missing proteins with ≥2 non-nested peptides were validated by careful spectral matching.

Background: AQP2 water channel is critical for urinary concentration in the kidney. Interestingly, AQP2 is abundantly excreted in the urine as extracellular vesicles (EVs), which is known to be a useful biomarker for water-balance disorders although the character of AQP2-enriched EVs is poorly understood including water channel function., Methods: Human urine EVs were obtained by a differential centrifugation method. AQP2-bearing EVs were isolated by immunoprecipitation with an AQP2-specific antibody, and the proteins in the EVs were analyzed by LC-MS/MS proteomic analysis. Osmotic water permeability (Pf) of the AQP2-rich EVs was measured by a stopped-flow method monitoring scattered light intensity in response to outwardly directed osmotic gradient., Results: Sequential centrifugation of human urine showed that AQP2 was present predominantly (80%) in low-density EVs (160,000 g), whereas negligible amount in high-density EVs (17,000 g). Proteomic analysis of the AQP2-bearing EVs identified 137 proteins, mostly in the endosome pathway, including the components of ESCRT (endosomal sorting complex required transporter)-I, II, III. Pf value of the 160,000 g EVs was 4.75 ± 0.38 × 10 -4 cm s -1 (mean ± SE) with the activation energy of 3.51 kcal mol -1 which was inhibited with 0.3 mM HgCl 2 by 63%, suggesting a channel-mediated water transport. Moreover, Pf value showed a significant correlation with the abundance of AQP2 protein in EVs., Conclusion: Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.

Oral squamous cell carcinomas (SCCs) are frequently associated with pre-invasive lesions including carcinoma in-situ (CIS), and CISs further form lateral interfaces against surrounding normal or dysplastic epithelia (ND). At the interface where keratin (K) 17 positive (+) SCC/CIS cells are in contact with K13+ ND cells, "cell competition" must be evoked between two such different cell types. Thus, the aim of this study was to characterize the histopathology of the SCC/CIS-ND interface and to determine protein profiles around the interface by proteomics. A total of 112 lateral interfaces were collected from 55 CIS and 57 SCC foci, and they were investigated by immunohistochemistry and liquid chromatography-tandem mass spectrometry. The interfaces were morphologically classified into three types: vertical, oblique, and convex. There were several cellular changes characteristic to the interface, including apoptosis and hyaline bodies, which were more emphasized in SCC/CIS sides. The results suggested that ND cells were winners of cell competition against SCC/CIS cells. Then, the interfaces were divided into four vertical segments, and each segment was separately laser-microdissected from tissue sections with immunostaining for K13 or K17; the four segments included SCC/CIS away from (#1) or adjacent to (#2) the interface, and ND adjacent to (#3) or away from (#4) the interface. Proteome analyses revealed approximately 4000 proteins from SCC/CIS sides [#1 and #2] and 2800 proteins from ND sides [#3 and #4]. We quantitatively selected the top 25 proteins including ladinin-1 or interleukin-1 receptor antagonist protein, which were most contrastively increased or decreased in SCC/CIS or ND sides, respectively, and their specific immunohistochemical expression modes were confirmed in tissue sections as well as in cultured SCC cells. These molecules should be involved in the cellular crosstalk toward cell competition at the lateral interface of oral SCC/CIS and would be new candidates for histopathological distinction of oral malignancies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Major advancements have recently been made in mass spectrometry-based proteomics, yielding an increasing number of datasets from various proteomics projects worldwide. In order to facilitate the sharing and reuse of promising datasets, it is important to construct appropriate, high-quality public data repositories. jPOSTrepo (https://repository.jpostdb.org/) has successfully implemented several unique features, including high-speed file uploading, flexible file management and easy-to-use interfaces. This repository has been launched as a public repository containing various proteomic datasets and is available for researchers worldwide. In addition, our repository has joined the ProteomeXchange consortium, which includes the most popular public repositories such as PRIDE in Europe for MS/MS datasets and PASSEL for SRM datasets in the USA. Later MassIVE was introduced in the USA and accepted into the ProteomeXchange, as was our repository in July 2016, providing important datasets from Asia/Oceania. Accordingly, this repository thus contributes to a global alliance to share and store all datasets from a wide variety of proteomics experiments. Thus, the repository is expected to become a major repository, particularly for data collected in the Asia/Oceania region., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Biology and Disease-driven Human Proteome Project (B/D-HPP) is aimed at supporting and enhancing the broad use of state-of-the-art proteomic methods to characterize and quantify proteins for in-depth understanding of the molecular mechanisms of biological processes and human disease. Based on a foundation of the pre-existing HUPO initiatives begun in 2002, the B/D-HPP is designed to provide standardized methods and resources for mass spectrometry and specific protein affinity reagents and facilitate accessibility of these resources to the broader life sciences research and clinical communities. Currently there are 22 B/D-HPP initiatives and 3 closely related HPP resource pillars. The B/D-HPP groups are working to define sets of protein targets that are highly relevant to each particular field to deliver relevant assays for the measurement of these selected targets and to disseminate and make publicly accessible the information and tools generated. Major developments are the 2016 publications of the Human SRM Atlas and of "popular protein sets" for six organ systems. Here we present the current activities and plans of the BD-HPP initiatives as highlighted in numerous B/D-HPP workshops at the 14th annual HUPO 2015 World Congress of Proteomics in Vancouver, Canada., Competing Interests: The authors declare no competing financial interest.

To understand functions and diseases of urinary bladder, it is important to define its molecular constituents and their roles in urinary bladder biology. Here, we performed genome-wide deep RNA sequencing analysis of human urinary bladder samples and identified genes up-regulated in the urinary bladder by comparing the transcriptome data to those of all other major human tissue types. 90 protein-coding genes were elevated in the urinary bladder, either with enhanced expression uniquely in the urinary bladder or elevated expression together with at least one other tissue (group enriched). We further examined the localization of these proteins by immunohistochemistry and tissue microarrays and 20 of these 90 proteins were localized to the whole urothelium with a majority not yet described in the context of the urinary bladder. Four additional proteins were found specifically in the umbrella cells (Uroplakin 1a, 2, 3a, and 3b), and three in the intermediate/basal cells (KRT17, PCP4L1 and ATP1A4). 61 of the 90 elevated genes have not been previously described in the context of urinary bladder and the corresponding proteins are interesting targets for more in-depth studies. In summary, an integrated omics approach using transcriptomics and antibody-based profiling has been used to define a comprehensive list of proteins elevated in the urinary bladder.

OFFGEL fractionation of mouse kidney protein lysate and its tryptic peptide digest has been examined in this study for better understanding the differences between protein and peptide fractionation methods and attaining maximum recruitment of this modern methodology for in-depth proteomic analysis. With the same initial protein/peptide load for both fractionation methods, protein OFFGEL fractionation showed a preponderance in terms of protein identification, fractionation efficiency, and focusing resolution, while peptide OFFGEL was better in recovery, number of peptide matches, and protein coverage. This result suggests that the protein fractionation method is more suitable for shotgun analysis while peptide fractionation suits well quantitative peptide analysis [isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT)]. Taken together, utilization of the advantages of both fractionation approaches could be attained by coupling both methods to be applied on complex biological tissue. A typical result is shown in this article by identification of 8262 confident proteins of whole mouse kidney under stringent condition. We therefore consider OFFGEL fractionation as an effective and efficient addition to both label-free and quantitative label proteomics workflow.

Formalin-fixed paraffin-embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin-induced alternations on a proteome-wide level. We compared LC-MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2-6% of all peptide-spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Embryonic stem cells (ESCs) have the ability to self-renew indefinitely and they can give unlimited source of cells and tissues for cellular therapies. Recently, the natriuretic peptide receptor A (NPR-A) has been recognized as an important regulator for the self-renewal of ESCs. To gain insights into possible novel mechanisms involved in NPR-A pathway that presumably regulates self-renewal and survival of ESCs, we utilized a comprehensive label-free proteomics technology in our study. Targeting of NPR-A gene with small interfering RNA (siRNA) resulted in the inhibition of ESCs self-renewal. Coherently, quantitative label-free shotgun proteomic analysis identified differentially expressed proteins involved in several biological processes, including cell cycle regulation, cell proliferation, cell fate specification, and apoptosis. Interestingly, in addition to Oct4 Nanog, and Sox2, other proteins involved in ESCs self-renewal were down-regulated after NPR-A knockdown, such as heterogeneous nuclear ribonucleoprotein A2/B1 (ROA2), non-POU domain-containing octamer-binding protein (Nono), nucleoplasmin (Npm1), histone H2A type 1-B/E (histone H2A.2), SW1/SNF complex (Brg1), polycomb protein Suz12 (Suz12), and cyclin-dependent kinase 4 (Cdk4). Furthermore, several protein candidates involved in early differentiation and cell death were up-regulated or down-regulated as a result of NPR-A knockdown, including importin subunit alpha-4 (Impα4), importin-5 (Ipo5), H3 histones, core histone macro-H2A.1 (H2A.y), apurine/apyrimidine endonuclease 1 (Apex1), 78-kDa glucose-regulated protein (Grp78), and programmed cell death 5 (Pdcd5). Overall, these findings depict a comprehensive view to our understanding of the pathways involved in the role of NPR-A in maintaining ESC functions.

Purpose: Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples., Experimental Design: Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins., Results: Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins., Conclusions and Clinical Relevance: Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organization's organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for both prospective and retrospective biomarker discovery. Unlocking the proteomic profile of clinicopathological FFPE tissues is a critically essential step for annotating clinical findings and predicting biomarkers for ultimate disease prognosis and therapeutic follow-up., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC-MS alignment based approach, we compared wild-type and AQP8 knockout mice colon proteomes. Shared result between both experiments revealed down-regulation of α-amylase 2 in AQP8-deleted mice model. Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in α-amylase 2 regulation. Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating α-amylase 2 synthesis by colonic epithelial cells as well. Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts.

Background: Mutation of the epidermal growth factor receptor (EGFR) results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling., Methodology/principal Findings: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992), one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics., Conclusions/significance: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling.

Databases which are useful for proteomic analysis of human kidney tissue and urine have been discussed in this article. Integration of the gene-centric and protein-centric general databases with those of human kidney tissue and urine proteomes may open a new window for research in nephrology. Proteins present in the kidney and urine provide basic tools for investigation of kidney function and disease. By comparing such databases between the healthy and diseased populations, we may be able to identify the following: proteins involved in the development of renal disease, proteins involved in progression of CKD, or new biomarker candidate proteins for either the development of renal disease or the progression of CKD., (Copyright © 2010 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Clinical proteomics has yielded some early positive results-the identification of potential disease biomarkers-indicating the promise for this analytical approach to improve the current state of the art in clinical practice. However, the inability to verify some candidate molecules in subsequent studies has led to skepticism among many clinicians and regulatory bodies, and it has become evident that commonly encountered shortcomings in fundamental aspects of experimental design mainly during biomarker discovery must be addressed in order to provide robust data. In this Perspective, we assert that successful studies generally use suitable statistical approaches for biomarker definition and confirm results in independent test sets; in addition, we describe a brief set of practical and feasible recommendations that we have developed for investigators to properly identify and qualify proteomic biomarkers, which could also be used as reporting requirements. Such recommendations should help put proteomic biomarker discovery on the solid ground needed for turning the old promise into a new reality.

With the completion or almost completion of genome sequences of many organisms in combination with the tremendous development of mass spectrometric analysis of proteins, several comprehensive proteomic studies, targeting whole organisms, body fluids, organs, tissues, cells, cellular organelles, or functional protein complexes, have produced valuable resources that can be shared and retrieved. In the present review, we provide current concept of construction of protein databases with special emphasis on high-throughput identification of protein using mass spectrometry, annotations, computational tools, and search engines to retrieve information of the identified proteins. We then update the current status of available protein databases of kidney and urine proteomes.

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

To understand renal functions and disease, it is important to define the molecular constituents of the various compartments of the kidney. Here, we used comparative transcriptomic analysis of all major organs and tissues in the human body, in combination with kidney tissue micro array based immunohistochemistry, to generate a comprehensive description of the kidney-specific transcriptome and proteome. A special emphasis was placed on the identification of genes and proteins that were elevated in specific kidney subcompartments. Our analysis identified close to 400 genes that had elevated expression in the kidney, as compared to the other analysed tissues, and these were further subdivided, depending on expression levels, into tissue enriched, group enriched or tissue enhanced. Immunohistochemistry allowed us to identify proteins with distinct localisation to the glomeruli (n = 11), proximal tubules (n = 120), distal tubules (n = 9) or collecting ducts (n = 8). Among the identified kidney elevated transcripts, we found several proteins not previously characterised or identified as elevated in kidney. This description of the kidney specific transcriptome and proteome provides a resource for basic and clinical research to facilitate studies to understand kidney biology and disease. [ABSTRACT FROM AUTHOR]

Background. Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney. [ABSTRACT FROM PUBLISHER]

Conventional sauna bathing may have some health benefits as it facilitates relaxing, detoxing and promoting metabolism. However, conventional sauna bathing at a high temperature may be harmful for the body by increasing the risk of heart failure. The nano-mist sauna has been developed to provide nano-size water particles at a lower temperature. Hence, nano-mist sauna bathing is expected to be useful for health without the risks that arise at high temperatures. In this study, we performed a comprehensive proteomics analysis of urine samples obtained from healthy volunteers before and after they had taken a sauna bath with nano-mist (n = 10) or with conventional mist (n = 10) daily for two weeks (4 groups). The average numbers of urine proteins identified by liquid chromatography-linked mass chromatography in each group varied from 678 to 753. Interestingly, the protein number was increased after sauna bathing both with nano-mist or with conventional mist. Quantitative analysis indicated that considerable numbers of proteins were obviously up-regulated, with more than two-folds in urine samples after the nano-mist sauna bathing. The KEGG pathway analysis showed significant stimulation of the lysosome pathway (p = 5.89E−6) after the nano-mist bathing, which may indicate the nano-mist sauna bathing promotes metabolism related to the lysosome pathway more efficiently than conventional mist sauna bathing and may provide more health benefits. [ABSTRACT FROM AUTHOR]

Since glomerular sclerosis frequently accompanies various glomerular diseases at the end stages, it is challenging to differentiate ubiquitous biological processes underlying this pathology from those critically involved in specific diseases. Furthermore, in-depth proteomic profile of human glomerular sclerosis remains limited. In this study, human glomeruli with intermediate (i-GS) and advanced (GS) sclerotic lesions, which were excluded from specific renal diseases and assumed to be aging-related, were laser captured from macroscopically normal cortex distant from urological carcinoma, and subjected to label-free quantitative proteomic analysis. We explicate an evident increase of membrane attack complex in i-GS and GS with an up-going tendency, which is accompanied by increasing of inhibitory regulators of alternative and terminal pathways. GO annotation and IPA pathway analysis agree to these results. Proteomic findings are validated by immunohistochemical studies which indicate that alternative and terminal pathways are positively involved in the glomerular sclerosis seen in distinct renal diseases. Furthermore, proteomic analysis also demonstrates remarkable increases of complement factor B in GS and TGF-ß1 in both GS and i-GS. Identification of complement factor B implicates that on-site activation of alternative pathway may occur in injured glomeruli and stepwise increase of TGF-ß1 suggests its contribution to the progression of glomerulosclerosis. Biological significance This study provides in-depth quantitative proteomic profiles of human glomeruli with intermediate and advanced sclerotic lesions. It reveals that the over-expression of alternative and terminal pathway components is significantly involved in human glomerulosclerosis seen in distinct renal diseases. Proteomic identification of the increased TGF-ß1 provides supporting evidence for the role of podocyte apoptosis leading to human glomerulosclerosis. [ABSTRACT FROM AUTHOR]

Abstract: Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ∼24% virulence determinants and toxins, ∼17% involved in carbohydrate metabolism, ∼14% involved in environmental stress, and ∼12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting. [Copyright &y& Elsevier]

Highlights: [•] Heat shock protein (HSP) A9 is a novel interaction molecule with podoplanin. [•] HSPA9 is secreted by oral squamous cell carcinoma cells. [•] HSPA9 interacts with podoplanin on the cell membrane. [•] HSPA9 and podoplanin cooperate in invasion of oral squamous cell carcinoma cells. [ABSTRACT FROM AUTHOR]

Abstract: European community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) clone remains a striking pathogenic clone spreading in European and Mediterranean countries. Since analysis of the secretome produced from this clone by proteomics could provide a comprehensive picture of both core exoproteins as well as virulence factors, we applied two proteomic approaches, pre-fractionation of proteins on SDS-PAGE followed by in-gel trypsin digestion, and in-solution trypsin-digestion followed by off-line SCX fractionation, both of which were coupled with LC–MS/MS analyses. A total of 174 distinct proteins were identified with a high-confidence. Functional classification of these identified proteins resulted in16.09% of protein synthesis, 13.79% of virulence, 6.89% of toxin, and 17.24% of unknown function. Prediction of their cellular localizations revealed 18.39% in extracellular space, 36.20% in cytoplasm, 5.17% in cytoplasmic membranes, 6.89% in cell wall, 1.14% in multiple localizations, and 32.18% in unknown localization. Among them, 52% proteins were predicted to be secreted through signal peptide-independent pathways. Most notably, the expression of some proteins such as enterotoxins U and B were identified for the first time in this clone. [Copyright &y& Elsevier]

Abstract: In the field of bottom-up proteomics, heavy contamination of human keratins could hinder the comprehensive protein identification, especially for the detection of low abundance proteins. In this study, we examined the keratin contamination in the four major experimental procedures in gel-based proteomic analysis including gel preparation, gel electrophoresis, gel staining, and in-gel digestion. We found that in-gel digestion procedure might be of importance corresponding to keratin contaminants compared to the other three ones. The human keratin contamination was reduced significantly by using an electrostatic eliminator during in-gel digestion, suggesting that static electricity built up on insulated experimental materials might be one of the essential causes of keratin contamination. We herein proposed a series of methods for improving experimental conditions and sample treatment in order to minimize the keratin contamination in an economical and practical way. [Copyright &y& Elsevier]

Abstract: Aquaporin (AQP) family plays a fundamental role in transmembrane water and small solutes movement. Within this family, aquaporin 8 (AQP8), showed to be widely distributed in the digestive system especially colon. To investigate the possible protein alterations involved in AQP8 regulation and trafficking, we extensively compared between wild type and AQP8 knockout mouse colon using semi-quantitative fluorescence- stained two dimensional gel electrophoresis (2-DE) coupled with nano LC-Ms/Ms. Our analysis revealed identification and regulation of 21 proteins, most notably, actin-related family which suggests its possible involvement in regulating AQP8 secretory vesicles migration to be integrated as a cell membrane protein. [Copyright &y& Elsevier]