A prospective, proteomics study identified potential biomarkers of encapsulating peritoneal sclerosis in peritoneal effluent.

Encapsulating peritoneal sclerosis (EPS) is a potentially devastating complication of peritoneal dialysis (PD). Diagnosis is often delayed due to the lack of effective and accurate diagnostic tools. We therefore examined peritoneal effluent for potential biomarkers that could predict or confirm the diagnosis of EPS and would be valuable in stratifying at-risk patients and driving appropriate interventions. Using prospectively collected samples from the Global Fluid Study and a cohort of Greek PD patients, we utilized 2D SDSPAGE/ MS and iTRAQ to identify changes in the peritoneal effluent proteome from patients diagnosed with EPS and controls matched for treatment exposure. We employed a combinatorial peptide ligand library to compress the dynamic range of protein concentrations to aid identification of low-abundance proteins. In patients with stable membrane function, fibrinogen γ-chain and heparan sulphate proteoglycan core protein progressively increased over time on PD. In patients who developed EPS, collagen-α1(I), γ-actin and Complement factors B and I were elevated up to five years prior to diagnosis. Orosomucoid-1 and a2-HS-glycoprotein chain-B were elevated about one year before diagnosis, while apolipoprotein A-IV and α1-antitrypsin were decreased compared to controls. Dynamic range compression resulted in an increased number of proteins detected with improved resolution of protein spots, compared to the full fluid proteome. Intelectin-1, dermatopontin, gelsolin, and retinol binding protein-4 were elevated in proteome-mined samples from patients with EPS compared to patients that had just commenced peritoneal dialysis. Thus, prospective analysis of peritoneal effluent uncovered proteins indicative of inflammatory and pro-fibrotic injury worthy of further evaluation as diagnostic/prognostic markers.

Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

Proteomic analysis of IgE-mediated secretion by LAD2 mast cells.

The LAD2 cell line is a relatively recent addition to the range of mast cell analogues and is of particular importance as it is the only human analogue which can be stimulated to degranulate in an IgE-dependent manner. Mast cells are tissue-based effector cells which have historically been shown to play an important role in the adaptive immune response, though there is now gathering evidence of their significance as a component of the innate immune system. These functions can be attributed to the ability of mast cells to regulate secretion of a wide variety of potent biologically active mediators through immediate and delayed responses. This well-orchestrated secretory mechanism of the mast cell makes it an ideal model in which to study this event. In this investigation, two-dimensional electrophoresis was employed as part of the proteomic characterization of the LAD2 human mast cell line, focusing in particular on a global analysis of membrane protein relocation after an IgE-mediated stimulatory event. This investigation has identified six membrane-associated protein spots which became phosphorylated upon IgE-mediated activation, 31 protein spots which displayed consistent recruitment to the membrane fraction, and three which were consistently lost from the soluble fraction. The scenario which emerges reveals a series of substantial changes which affect every compartment of the cell, providing evidence for a coordinated response to a secretory stimulus.

Peptide aptamers in label-free protein detection: 2. Chemical optimization and detection of distinct protein isoforms.

The early detection and diagnosis of cancer lies central to successful treatment and improved patient outcome. Current techniques are limited by the nature of the biological receptor and the assays available. This paper reports the use of novel biological probes, peptide aptamers, in detecting cyclin-dependent protein kinases (CDKs) whose activity is important in proliferating and cancerous cells. We describe, specifically, the optimization of an orientated peptide aptamer surface and its utilization in establishing a highly specific, low-nanomolar sensitive, detection protocol for the active form of CDK2. In comparing target binding affinity of two different aptamers (pep6 and pep9), both constructed through the insertion of peptide sequences into the surface of a scaffold protein, one was observed to be consistently more effective. Significantly, the pep9 aptamers were able to detect subtle changes in the conformation of CDK2 associated with activation of its catalytic activity that may be caused by the phosphorylation of a single amino acid (threonine 160). A typical response toward the inactive form of CDK2 was in the range of 0.5-2% of the binding of the active form of CDK2 in the concentration range from 2 to 20 nM. Although antibodies are occasionally able to recognize conformations in their targets, this is the first time that a nonantibody protein probe has been used to detect an active protein isoform. Because peptide aptamers are usually raised against full-length proteins, this raises the possibility that peptide aptamers will be able to extend the repertoire of probes that recognize protein conformations, post-translational modifications (PTMs), or conformations stabilized by PTMs.