title: Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine.
abstract: Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.
authors.affiliation: Biomedical Research Foundation Academy of Athens, Biotechnology Division, Athens, Greece; Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Szymon
authors.initials: S
authors.lastname: Filip
authors.affiliation: Biomedical Research Foundation Academy of Athens, Biotechnology Division, Athens, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Vougas
authors.affiliation: Biomedical Research Foundation Academy of Athens, Biotechnology Division, Athens, Greece.
authors.forename: Jerome
authors.initials: J
authors.lastname: Zoidakis
authors.affiliation: Biomedical Research Foundation Academy of Athens, Biotechnology Division, Athens, Greece; Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Latosinska
authors.affiliation: University of Glasgow Institute of Cardiovascular and Medical Sciences, Glasgow, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Mullen
authors.affiliation: Ss. Cyril and Methodius University in Skopje, Nephrology Department, Skopje, Former Yugoslav Republic of Macedonia.
authors.forename: Goce
authors.initials: G
authors.lastname: Spasovski
authors.affiliation: University of Glasgow Institute of Cardiovascular and Medical Sciences, Glasgow, United Kingdom; Mosaiques Diagnostics GmbH, Hannover, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Mischak
authors.affiliation: Biomedical Research Foundation Academy of Athens, Biotechnology Division, Athens, Greece.
authors.forename: Antonia
authors.initials: A
authors.lastname: Vlahou
authors.affiliation: University Hospital RWTH Aachen, Institute for Molecular Cardiovascular Research, Aachen, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Jankowski
mesh_terms: D015415:Biomarkers / Q000652:urine; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000378:metabolism / Q000652:urine; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D051436:Renal Insufficiency, Chronic / Q000378:metabolism / Q000652:urine
chemical_list: D015415:Biomarkers; D010455:Peptides; D020543:Proteome