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BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by abnormal protein aggregation in the motor neurons. Present and earlier proteomic studies to characterize peptides in cerebrospinal fluid (CSF) associated with motoneuron pathology did not target low molecular weight proteins and peptides. We hypothesized that specific changes in CSF peptides or low molecular weight proteins are significantly altered in ALS, and that these changes may support deciphering molecular pathophysiology and even guide approaches towards therapeutic interventions.MethodsCerebrospinal fluid (CSF) from 50 ALS patients and 50 non-ALS controls was collected, centrifuged immediately after collection, aliquoted into polypropylene test tubes, frozen within 30-40 min after the puncture, and stored at -80°C until use. Peptides were sequenced using capillary electrophoresis or liquid chromatography/mass spectrometry (CE-MS/MS or LC-MS/MS).FindingsIn the CSF of 50 patients and 50 non-ALS controls 33 peptides were found, of which 14 could be sequenced using a non-lytic single-pot proteomic detection method, CE/MS. ALS deregulated peptides vs. controls included Integral membrane protein 2B, Neurosecretory protein VGF, Osteopontin, Neuroendocrine protein 7B2 (Secretogranin-V), EGF-containing fibulin-like extracellular matrix protein 1, Xylosyltransferase 1 XT-1, Chromogranin-A, Superoxide dismutase SOD-1, Secretogranin-1 (Chromogranin B), NR2F2 Nuclear Receptor Subfamily 2 Group F Member 2 and Collagen alpha-1(VII) chain.InterpretationMost striking deregulations in CSF from ALS patients were found in VGF, Osteopontin, SOD-1 and EFEMP1 peptides. No associations of disease severity, duration and region of onset with sequenced peptides were found.

Chronic kidney disease (CKD) is characterized by the loss of kidney function. The molecular mechanisms underlying the development and progression of CKD are still not fully understood. Among others, the urinary peptidome has been extensively studied, with several urinary peptides effectively detecting disease progression. However, their link to proteolytic events has not been made yet. This study aimed to predict the proteases involved in the generation of CKD-associated urinary excreted peptides in a well-matched (for age, sex, lack of heart disease) case-control study. The urinary peptide profiles from CKD (n = 241) and controls (n = 240) were compared and statistically analyzed. The in-silico analysis of the involved proteases was performed using Proteasix and proteases activity was predicted based on the abundance changes of the associated peptides. Predictions were cross-correlated to transcriptomics datasets by using the Nephroseq database. Information on the respective protease inhibitors was also retrieved from the MEROPS database. Totally, 303 urinary peptides were significantly associated with CKD. Among the most frequently observed were fragments of collagen types I, II and III, uromodulin, albumin and beta-2-microglobulin. Proteasix predicted 16 proteases involved in their generation. Through investigating CKD-associated transcriptomics datasets, several proteases are highlighted including members of matrix metalloproteinases (MMP7, MMP14) and serine proteases (PCSK5); laying the foundation for further studies towards elucidating their role in CKD pathophysiology.

Parental investment in Arapaima gigas includes nest building and guarding, followed by a care provision when a cephalic fluid is released from the parents' head to the offspring. This fluid has presumably important functions for the offspring but so far its composition has not been characterised. In this study the proteome and peptidome of the cephalic secretion was studied in parental and non-parental fish using capillary electrophoresis coupled to mass spectrometry (CE-MS) and GeLC-MS/MS analyses. Multiple comparisons revealed 28 peptides were significantly different between males and parental males (PC-males), 126 between females and parental females (PC-females), 51 between males and females and 9 between PC-males and PC-females. Identification revealed peptides were produced in the inner ear (pcdh15b), eyes (tetraspanin and ppp2r3a), central nervous system (otud4, ribeye a, tjp1b and syn1) among others. A total of 422 proteins were also identified and gene ontology analysis revealed 28 secreted extracellular proteins. From these, 2 hormones (prolactin and stanniocalcin) and 12 proteins associated to immunological processes (serotransferrin, α-1-antitrypsin homolog, apolipoprotein A-I, and others) were identified. This study provides novel biochemical data on the lateral line fluid which will enable future hypotheses-driven experiments to better understand the physiological roles of the lateral line in chemical communication.

To analyze the protein profile of human vitreous of untreated patients with retinal vein occlusion (RVO).Sixty-eight vitreous humor (VH) samples (44 from patients with treatment naïve RVO, 24 controls with idiopathic floaters) were analyzed in this clinical-experimental study using capillary electrophoresis coupled to mass spectrometer and tandem mass spectrometry. To define potential candidate protein markers of RVO, proteomic analysis was performed on RVO patients (n = 30) and compared with controls (n = 16). To determine validity of potential biomarker candidates in RVO, receiver operating characteristic (ROC) was performed by using proteome data of independent RVO (n = 14) and control samples (n = 8).Ninety-four different proteins (736 tryptic peptides) could be identified. Sixteen proteins were found to be significant when comparing RVO and control samples (P = 1.43E-05 to 4.48E-02). Five proteins (Clusterin, Complement C3, Ig lambda-like polypeptide 5 (IGLL5), Opticin and Vitronectin), remained significant after using correction for multiple testing. These five proteins were also detected significant when comparing subgroups of RVO (central RVO, hemi-central RVO, branch RVO) to controls. Using independent samples ROC-Area under the curve was determined proving the validity of the results: Clusterin 0.884, Complement C3 0.955, IGLL5 1.000, Opticin 0.741, Vitronectin 0.786. In addition, validation through ELISA measurements was performed.The results of the study reveal that the proteomic composition of VH differed significantly between the patients with RVO and the controls. The proteins identified may serve as potential biomarkers for pathogenesis induced by RVO.

BackgroundThere is absence of specific biomarkers and an incomplete understanding of the pathophysiology of exudative age-related macular degeneration (AMD).Methods and findingsEighty-eight vitreous samples (73 from patients with treatment naïve AMD and 15 control samples from patients with idiopathic floaters) were analyzed with capillary electrophoresis coupled to mass spectrometry in this retrospective case series to define potential candidate protein markers of AMD. Nineteen proteins were found to be upregulated in vitreous of AMD patients. Most of the proteins were plasma derived and involved in biological (ion) transport, acute phase inflammatory reaction, and blood coagulation. A number of proteins have not been previously associated to AMD including alpha-1-antitrypsin, fibrinogen alpha chain and prostaglandin H2-D isomerase. Alpha-1-antitrypsin was validated in vitreous of an independent set of AMD patients using Western blot analysis. Further systems biology analysis of the data indicated that the observed proteomic changes may reflect upregulation of immune response and complement activity.ConclusionsProteome analysis of vitreous samples from patients with AMD, which underwent an intravitreal combination therapy including a core vitrectomy, steroids and bevacizumab, revealed apparent AMD-specific proteomic changes. The identified AMD-associated proteins provide some insight into the pathophysiological changes associated with AMD.

National Kidney Foundation CKD staging has allowed uniformity in studies on CKD. However, early diagnosis and predicting progression to end stage renal disease are yet to be improved. Seventy six patients with different levels of CKD, including outpatients and dialysed patients were studied for transcriptome, metabolome and proteome description. High resolution urinary proteome analysis was blindly performed in the 53 non-anuric out of the 76 CKD patients. In addition to routine clinical parameters, CKD273, a urinary proteomics-based classifier and its peptides were quantified. The baseline values were analyzed with regard to the clinical parameters and the occurrence of death or renal death during follow-up (3.6 years) as the main outcome measurements. None of the patients with CKD2730.55. Unsupervised clustering analysis of the CKD273 peptides separated the patients into two main groups differing in CKD associated parameters. Among the 273 biomarkers, peptides derived from serum proteins were relatively increased in patients with lower glomerular filtration rate, while collagen-derived peptides were relatively decreased (p

BACKGROUND: Extensive prostate specific antigen screening for prostate cancer generates a high number of unnecessary biopsies and over-treatment due to insufficient differentiation between indolent and aggressive tumours. We hypothesized that seminal plasma is a robust source of novel prostate cancer (PCa) biomarkers with the potential to improve primary diagnosis of and to distinguish advanced from indolent disease. METHODOLOGY/PRINCIPAL FINDINGS: In an open-label case/control study 125 patients (70 PCa, 21 benign prostate hyperplasia, 25 chronic prostatitis, 9 healthy controls) were enrolled in 3 centres. Biomarker panels a) for PCa diagnosis (comparison of PCa patients versus benign controls) and b) for advanced disease (comparison of patients with post surgery Gleason score 7) were sought. Independent cohorts were used for proteomic biomarker discovery and testing the performance of the identified biomarker profiles. Seminal plasma was profiled using capillary electrophoresis mass spectrometry. Pre-analytical stability and analytical precision of the proteome analysis were determined. Support vector machine learning was used for classification. Stepwise application of two biomarker signatures with 21 and 5 biomarkers provided 83% sensitivity and 67% specificity for PCa detection in a test set of samples. A panel of 11 biomarkers for advanced disease discriminated between patients with Gleason score 7 and organ-confined (

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87-89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots.

Treatment options for autosomal dominant polycystic kidney disease (ADPKD) will likely become available in the near future, hence reliable diagnostic and prognostic biomarkers for the disease are strongly needed. Here, we aimed to define urinary proteomic patterns in ADPKD patients, which aid diagnosis and risk stratification. By capillary electrophoresis online coupled to mass spectrometry (CE-MS), we compared the urinary peptidome of 41 ADPKD patients to 189 healthy controls and identified 657 peptides with significantly altered excretion, of which 209 could be sequenced using tandem mass spectrometry. A support-vector-machine based diagnostic biomarker model based on the 142 most consistent peptide markers achieved a diagnostic sensitivity of 84.5% and specificity of 94.2% in an independent validation cohort, consisting of 251 ADPKD patients from five different centers and 86 healthy controls. The proteomic alterations in ADPKD included, but were not limited to markers previously associated with acute kidney injury (AKI). The diagnostic biomarker model was highly specific for ADPKD when tested in a cohort consisting of 481 patients with a variety of renal and extrarenal diseases, including AKI. Similar to ultrasound, sensitivity and specificity of the diagnostic score depended on patient age and genotype. We were furthermore able to identify biomarkers for disease severity and progression. A proteomic severity score was developed to predict height adjusted total kidney volume (htTKV) based on proteomic analysis of 134 ADPKD patients and showed a correlation of r = 0.415 (p

Metabolic syndrome can induce chronic kidney disease in humans. Genetically engineered mice on a C57BL/6 background are highly used for mechanistic studies. Although it has been shown that metabolic syndrome induces cardiovascular lesions in C57BL/6 mice, in depth renal phenotyping has never been performed. Therefore in this study we characterized renal function and injury in C57BL/6 mice with long-term metabolic syndrome induced by a high fat and fructose diet (HFFD). C57BL/6 mice received an 8 months HFFD diet enriched with fat (45% energy from fat) and drinking water enriched with fructose (30%). Body weight, food/water consumption, energy intake, fat/lean mass ratio, plasma glucose, HDL, LDL, triglycerides and cholesterol levels were monitored. At 3, 6 and 8 months, renal function was determined by inulin clearance and measure of albuminuria. At sacrifice, kidneys and liver were collected. Metabolic syndrome in C57BL/6 mice fed a HFFD was observed as early 4 weeks with development of type 2 diabetes at 8 weeks after initiation of diet. However, detailed analysis of kidney structure and function showed only minimal renal injury after 8 months of HFFD. HFFD induced moderate glomerular hyperfiltration (436,4 µL/min vs 289,8 µL/min; p-value=0.0418) together with a 2-fold increase in albuminuria only after 8 months of HFFD. This was accompanied by a 2-fold increase in renal inflammation (p-value=0.0217) but without renal fibrosis or mesangial matrix expansion. In addition, electron microscopy did not show alterations in glomeruli such as basal membrane thickening and foot process effacement. Finally, comparison of the urinary peptidome of these mice with the urinary peptidome from humans with diabetic nephropathy also suggested absence of diabetic nephropathy in this model. This study provides evidence that the HFFD C57BL/6 model is not the optimal model to study the effects of metabolic syndrome on the development of diabetic kidney disease.

Representative animal models for diabetes-associated vascular complications are extremely relevant in assessing potential therapeutic drugs. While several rodent models for type 2 diabetes (T2D) are available, their relevance in recapitulating renal and cardiovascular features of diabetes in man is not entirely clear. Here we evaluate at the molecular level the similarity between Zucker diabetic fatty (ZDF) rats, as a model of T2D-associated vascular complications, and human disease by urinary proteome analysis. Urine analysis of ZDF rats at early and late stages of disease compared to age- matched LEAN rats identified 180 peptides as potentially associated with diabetes complications. Overlaps with human chronic kidney disease (CKD) and cardiovascular disease (CVD) biomarkers were observed, corresponding to proteins marking kidney damage (eg albumin, alpha-1 antitrypsin) or related to disease development (collagen). Concordance in regulation of these peptides in rats versus humans was more pronounced in the CVD compared to the CKD panels. In addition, disease-associated predicted protease activities in ZDF rats showed higher similarities to the predicted activities in human CVD. Based on urinary peptidomic analysis, the ZDF rat model displays similarity to human CVD but might not be the most appropriate model to display human CKD on a molecular level.

Improved biomarkers of acute nephrotoxicity are coveted by the drug development industry, regulatory agencies, and clinicians. In an effort to identify such biomarkers, urinary peptide profiles of rats treated with two different nephrotoxins were investigated. 493 marker candidates were defined that showed a significant response to cis-platin comparing a cis-platin treated cohort to controls. Next, urine samples from rats that received three consecutive daily doses of 150 or 300 mg/kg gentamicin were examined. 557 potential biomarkers were initially identified; 108 of these gentamicin-response markers showed a clear temporal response to treatment. 39 of the cisplatin-response markers also displayed a clear response to gentamicin. Of the combined 147 peptides, 101 were similarly regulated by gentamicin or cis-platin and 54 could be identified by tandem mass spectrometry. Most were collagen type I and type III fragments up-regulated in response to gentamicin treatment. Based on these peptides, classification models were generated and validated in a longitudinal study. In agreement with histopathology, the observed changes in classification scores were transient, initiated after the first dose, and generally persistent over a period of 10-20 days before returning to control levels. The data support the hypothesis that gentamicin-induced renal toxicity up-regulates protease activity, resulting in an increase in several specific urinary collagen fragments. Urinary proteomic biomarkers identified here, especially those common to both nephrotoxins, may serve as a valuable tool to investigate potential new drug candidates for the risk of nephrotoxicity.

Female patients affected by Fabry disease, an X-linked lysosomal storage disorder, exhibit a wide spectrum of symptoms, which renders diagnosis, and treatment decisions challenging. No diagnostic test, other than sequencing of the alpha-galactosidase A gene, is available and no biomarker has been proven useful to screen for the disease, predict disease course and monitor response to enzyme replacement therapy. Here, we used urine proteomic analysis based on capillary electrophoresis coupled to mass spectrometry and identified a biomarker profile in adult female Fabry patients. Urine samples were taken from 35 treatment-naïve female Fabry patients and were compared to 89 age-matched healthy controls. We found a diagnostic biomarker pattern that exhibited 88.2% sensitivity and 97.8% specificity when tested in an independent validation cohort consisting of 17 treatment-naïve Fabry patients and 45 controls. The model remained highly specific when applied to additional control patients with a variety of other renal, metabolic and cardiovascular diseases. Several of the 64 identified diagnostic biomarkers showed correlations with measures of disease severity. Notably, most biomarkers responded to enzyme replacement therapy, and 8 of 11 treated patients scored negative for Fabry disease in the diagnostic model. In conclusion, we defined a urinary biomarker model that seems to be of diagnostic use for Fabry disease in female patients and may be used to monitor response to enzyme replacement therapy.

The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D).Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92-95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81-94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81-88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed.These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling.