In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.
Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P < 0.01). 2-DE was essential in fractionation of the complex urine proteome, producing a reference map that included 20 proteins. Cauxin was the most abundant protein in urine of healthy cats. Several protease inhibitors and transport proteins that derive from plasma were also identified, including alpha-2-macroglobulin, albumin, transferrin, haemopexin and haptoglobin. There was differential expression of 27 spots between healthy and CKD samples (P < 0.05) and 13 proteins were unambiguously identified. In particular, increased expression of retinol-binding protein, cystatin M and apolipoprotein-H associated with decreased expression of uromodulin and cauxin confirmed tubular damage in CKD cats suggesting that these proteins are candidate biomarkers.
BackgroundMedication-overuse headache (MOH) is a chronic headache condition that results from the overuse of analgesics drugs, triptans, or other antimigraine compounds. The epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity, particularly in chronic patients. The aim of this work was to confirm and extend the results obtained from a previous study, in which we analyzed the urinary proteome of 3 MOH patients groups: non-steroidal anti-inflammatory drugs (NSAIDs), triptans and mixtures abusers, in comparison with non-abusers individuals (controls).MethodsIn the present work we employed specialized proteomic techniques, namely two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS), and the innovative Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS), to discover characteristic proteomic profiles associated with MOH condition.ResultsBy 2-DE and MS analysis we identified 21 over-excreted proteins in MOH patients, particularly in NSAIDs abusers, and the majority of these proteins were involved in a variety of renal impairments, as resulted from a literature search. Urine protein profiles generated by SELDI-TOF-MS analysis showed different spectra among groups. Moreover, significantly higher number of total protein spots and protein peaks were detected in NSAIDs and mixtures abusers.ConclusionsThese findings confirm the presence of alterations in proteins excretion in MOH patients. Analysis of urinary proteins by powerful proteomic technologies could lead to the discovery of early candidate biomarkers, that might allow to identify MOH patients prone to develop potential drug overuse-induced nephrotoxicity.
The unbalanced functional network of proteins involved could hinder adequate tissue response to pathogenic noxa. The study of periodontal pocket tissue proteomic profile would be crucial to better understand the pathogenesis of and the therapeutic strategies for periodontitis.
In previous works we showed the overexpression of some proteins in biological fluids from patients suffering chronic pain. In this proteomic study we analysed serum from a rat model of neuropathic pain obtained by the chronic constriction injury (CCI) of sciatic nerve, at two time intervals, 2 and 5 weeks after the insult, to find proteins involved in the expression or mediation of pain. Sham-operated and CCI rats were treated with saline or indomethacin. Two weeks after ligation, we identified three serum proteins overexpressed in CCI rats, two of which, alpha-1-macroglobulin and vitamin D-binding protein (VDBP), remained increased 5 weeks post-surgery; at this time interval, we found increased levels of further proteins, namely apolipoprotein A-I (APOA1), apolipoprotein E (APOE), prostaglandin-H2 D-isomerase (PTGDS) and transthyretin (TTR), that overlap the overexpressed proteins found in humans. Indomethacin treatment reversed the effects of ligation. The qPCR analysis showed that transcript levels of APOA1, APOE, PTGDS and VDBP were overexpressed in the lumbar spinal cord (origin of sciatic nerve), but not in the striatum (an unrelated brain region), of CCI rats treated with saline 5 weeks after surgery, demonstrating that the lumbar spinal cord is a possible source of these proteins.
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