Equalizer technology followed by DIGE‐based proteomics for detection of cellular proteins in artificial peritoneal dialysis effluents

Abstract

Peritoneal dialysis effluent (PDE) represents a rich pool of potential biomarkers for monitoring disease and therapy. Until now, proteomic studies have been hindered by the plasma-like composition of the PDE. Beads covered with a peptide library are a promising approach to remove high abundant proteins and concentrate the sample in one step. In this study, a novel approach for proteomic biomarker identification in PDEs consisting of a depletion and concentration step followed by 2D gel based protein quantification was established. To prove this experimental concept a model system of artificial PDEs was established by spiking unused peritoneal dialysis (PD) fluids with cellular proteins reflecting control conditions or cell stress. Using this procedure, we were able to reduce the amount of high abundant plasma proteins and concentrate low abundant proteins while preserving changes in abundance of proteins with cellular origin. The alterations in abundance of the investigated marker for cell stress, the heat shock proteins, showed similar abundance profiles in the artificial PDE as in pure cell culture samples. Our results demonstrate the efficacy of this system in detecting subtle changes in cellular protein expression triggered by unphysiological stress stimuli typical in PD, which could serve as biomarkers. Further studies using patients' PDE will be necessary to prove the concept in clinical PD and to assess whether this technique is also informative regarding enriching low abundant plasma derived protein biomarker in the PDE.

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures

“…Forty PDEs obtained from a prospective randomized controlled cross-over trial in 20 PD patients treated with standard or AlaGln-supplemented PD fluids were depleted and enriched using the CPLL approach previously developed in artificial PDE ( 20 ). Clinical results of a trial of this novel therapy were recently reported ( 27 ).…”

“…The PDE was processed immediately following the end of the dwell and aliquoted samples were stored at −80 °C until further analysis. The protocol was performed as previously reported ( 20 ) with minor adaptations for clinical samples. CPLL equalizer beads (ProteoMiner, BioRad, Hercules, CA) were prepared according to the manufacturer's protocol.…”

“…To minimize masking of low abundance candidate biomarker proteins by the background of high abundance proteins in PDE, we recently developed a depletion and enrichment strategy that was assessed in artificial PDE ( 20 ). Spiked-in aliquots of low abundance cellular proteins could be detected in PDE by two-dimensional difference gel electrophoresis (2D-DIGE) only by application of the combinatorial peptide ligand library (CPLL) technique.…”

Addition of Alanyl-Glutamine to Dialysis Fluid Restores Peritoneal Cellular Stress Responses – A First-In-Man Trial

“…Aliquots of the IPS were analyzed by 2D immunoblot as previously described [ 36 ]. The total protein pattern of the IPS was imaged on the membrane by fluorescence detection, and was used to guide alignment of immunoblot signals and 2D-DIGE quantification with the aid of the image warping capabilities of the Delta2D 4.3 software (Decodon, Greifswald, Germany).…”

Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2011–2014

“…Recently, Lichtenauer et al have established a novel approach for proteomic biomarker identification in peritoneal dialysis effluent (PDEs) using the equalizer technology followed by 2D-DIGE [37]. The experimental approach was proven using a model system for PDE that is further referred to as artificial PDE, which was established by spiking unused peritoneal dialysis fluids with cellular proteins reflecting control conditions or cell stress.…”