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      Patterns of Proteins Removed with High-Flux Membranes on High-Volume Hemodiafiltration Detected with a MultiDimensional LC-MS/MS Strategy

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          Background: Aim of this prospective crossover study was to identify the nature of the middle-molecular weight solutes removed during high-volume post-dilution HDF. Methods: The efficiency in removing small molecules, protein-bound and middle-molecular proteins was evaluated in 16 chronic dialysis patients on post-dilution HDF with two high-flux dialyzer membranes (Amembris and Polyamix). Multidimensional Protein Identification Technology (MudPIT) was employed to identify middle-molecular weight solutes in spent dialysate. Results: Efficiency of post-dilution HDF in removing solutes of different MW was high with both membranes, but higher with Amembris than with Polyamix. With MudPIT analysis, 277 proteins were identified in the dialysate fluids. Although the protein-removal pattern was similar among patients and tested membranes, the total and protein-specific peptide spectral count (mass spectrometric quantitation criteria) of most proteins were higher using the Amembris membrane. Conclusions: The MudPIT approach showed to be a powerful tool to identify a broad molecular weight spectrum of proteins removed with post-dilution HDF. Short Summary: Aim of this prospective crossover study was to analyze the hydraulic properties of two high-flux dialyzer membranes (Amembris and Polyamix) during high-volume, post-dilution HDF and to evaluate the influence of these properties on the removal of proteins and peptides using an in-depth analysis of the spent dialysate. For this analysis, a liquid chromatography tandem mass spectrometry approach called MudPIT (Multidimensional Protein Identification Technology) was used to identify the middle molecular weight solutes present in the spent dialysate of patients. The capability of post-dilution HDF in removing solutes of different MW was very high with both dialyzers, but higher with the Amembris membrane. The proteomic MudPIT approach showed to be a powerful tool to identify a wide molecular spectrum of proteins removed from blood during post-dilution HDF. These results may contribute to address research toward a better knowledge of uremic toxins and the balance between the intended and unintended removal of undesired and beneficial proteins next to identification of new target proteins as potential candidates for uremic toxicity. i 2014 S. Karger AG, Basel

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          Most cited references 17

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          Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

          We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.
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            Free p-cresylsulphate is a predictor of mortality in patients at different stages of chronic kidney disease.

            Uraemic toxins are considered to be emerging mortality risk factors in chronic kidney disease (CKD) patients. p-Cresol (a prototype protein-bound uraemic retention solute) has been shown to exert toxic effects in vitro. Recently, it has been demonstrated that p-cresol is present in plasma as its sulphate conjugate, p-cresylsulphate. The present study evaluated the distribution of free and total p-cresylsulphate and sought to determine whether these parameters were associated with vascular calcification, arterial stiffness and mortality risk in a cohort of CKD patients. One hundred and thirty-nine patients (mean +/- SD age: 67 +/- 12; males: 60%) at different stages of CKD (8% at Stage 2, 26.5% at Stage 3, 26.5% at Stage 4, 7% at Stage 5 and 32% at Stage 5D) were enrolled in this study. Baseline total and free p-cresylsulphate presented an inverse relationship with renal function and were significantly associated with vascular calcification. During the study period (mean follow-up period: 779 +/- 185 days), 38 patients died [including 22 from cardiovascular (CV) causes]. In crude survival analyses, free (but not total) p-cresylsulphate was shown to be a predictor of overall and CV death. Higher free p-cresylsulphate levels (>0.051 mg/100 mL; median) were associated with mortality independently of well-known predictors of survival such as age, vascular calcification, anaemia and inflammation. Serum levels of free and total p-cresylsulphate (the main in vivo circulating metabolites of p-cresol) were elevated in later CKD stages. However, only free p-cresylsulphate seems to be a predictor of survival in CKD.
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              An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics


                Author and article information

                Blood Purif
                Blood Purification
                S. Karger AG
                December 2014
                20 November 2014
                : 38
                : 2
                : 115-126
                aDepartment of Nephrology and Dialysis, Fondazione Orizzonte, Bolognini Hospital, Seriate, Italy; bDepartment of Analytical Chemistry, Ruhr University Bochum, Bochum, Germany; cDepartment of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia; dB. Braun Avitum AG, Melsungen, Germany
                Author notes
                *Luciano A. Pedrini, Department of Nephrology and Dialysis, Bolognini Hospital, IT-Seriate (Italy), E-Mail axl.pedrini@virgilio.it
                365745 Blood Purif 2014;38:115-126
                © 2014 S. Karger AG, Basel

                Open Access License: This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) ( http://www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 5, Tables: 5, Pages: 12
                Original Paper


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