Proteomics profiling of urine with surface enhanced laser desorption/ionization time of flight mass spectrometry

BACKGROUND: Urine consists of a complex mixture of peptides and proteins and therefore is an interesting source of biomarkers. Because of its high throughput capacity SELDI-TOF-MS is a proteomics technology frequently used in biomarker studies. We compared the performance of seven SELDI protein chip types for profiling of urine using standard chip protocols. RESULTS: Performance was assessed by determining the number of detectable peaks and spot to spot variation for the seven array types and two different matrices: SPA and CHCA. A urine sample taken from one healthy volunteer was applied in eight-fold for each chip type/matrix combination. Data were analyzed for total number of detected peaks (S/N > 5). Spot to spot variation was determined by calculating the average CV of peak intensities. In addition, an inventory was made of detectable peaks with each chip and matrix type. Also the redundancy in peaks detected with the different chip/matrix combinations was determined. A total of 425 peaks (136 non-redundant peaks) could be detected when combining the data from the seven chip types and the two matrices. Most peaks were detected with the CM10 chip with CHCA (57 peaks). The Q10 with CHCA (51 peaks), SEND (48 peaks) and CM10 with SPA (48 peaks) also performed well. The CM10 chip with CHCA also has the best reproducibility with an average CV for peak intensity of 13%. CONCLUSION: The combination of SEND, CM10 with CHCA, CM10 with SPA, IMAC-Cu with SPA and H50 with CHCA provides the optimal information from the urine sample with good reproducibility. With this combination a total of 217 peaks (71 non-redundant peaks) can be detected with CV's ranging from 13 to 26%, depending on the chip and matrix type. Overall, CM10 with CHCA is the best performing chip type

Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid

Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI) analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fluid when precautions are taken towards protein breakdown

Metabolomic Profiling for Identification of Novel Potential Biomarkers in Cardiovascular Diseases

Metabolomics involves the identification and quantification of metabolites present in a biological system. Three different approaches can be used: metabolomic fingerprinting, metabolic profiling, and metabolic footprinting, in order to evaluate the clinical course of a disease, patient recovery, changes in response to surgical intervention or pharmacological treatment, as well as other associated features. Characteristic patterns of metabolites can be revealed that broaden our understanding of a particular disorder. In the present paper, common strategies and analytical techniques used in metabolomic studies are reviewed, particularly with reference to the cardiovascular field

Urinary Kininogen-1 and Retinol binding protein-4 respond to Acute Kidney Injury: Predictors of patient prognosis?

Implementation of therapy for acute kidney injury (AKI) depends on successful prediction of individual patient prognosis. Clinical markers as serum creatinine (sCr) have limitations in sensitivity and early response. The aim of the study was to identify novel molecules in urine which show altered levels in response to AKI and investigate their value as predictors of recovery. Changes in the urinary proteome were here investigated in a cohort of 88 subjects (55 AKI patients and 33 healthy donors) grouped in discovery and validation independent cohorts. Patients'urine was collected at three time points: within the first 48 h after diagnosis(T1), at 7 days of follow-up(T2) and at discharge of Nephrology(T3). Differential gel electrophoresis was performed and data were confirmed by Western blot (WB), liquid chromatography/mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). Retinol binding protein 4 (RBP4) and kininogen-1 (KNG1) were found significantly altered following AKI. RBP4 increased at T1, and progressively decreased towards normalization. Maintained decrease was observed for KNG1 from T1. Individual patient response along time revealed RBP4 responds to recovery earlier than sCr. In conclusion, KNG1 and RBP4 respond to AKI. By monitoring RBP4, patient's recovery can be anticipated pointing to a role of RBP4 in prognosis evaluation.Funding: from Instituto de Salud Carlos III: FIS PI11/01401, PI13/01873, FIS IF08/3667-1, CP09/00229, PI13/00047, PI10/00624, ISCIII-RETIC REDinREN RD012/0021. FEDER funds, Comunidad de Madrid/CIFRA S2010/BMD-2378, Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO, IDCSalud (3371/002) and Fundación Conchita Rábago de Jiménez Díaz, Proteomic Facility from Universidad Complutense de Madrid-Fundación Parque Científico de Madrid (UCM-FPCM), Spain, a member of ProteoRed-ISCIII Network member of ProteoRed- ISCIII Networ

Plasma Molecular Signatures in Hypertensive Patients With Renin-Angiotensin System Suppression: New Predictors of Renal Damage and De Novo Albuminuria Indicators

Albuminuria is a risk factor strongly associated with cardiovascular disease, the first cause of death in the general population. It is well established that renin-angiotensin system suppressors prevent the development of new-onset albuminuria in naïf hypertensive patients and diminish its excretion, but we cannot forget the percentage of hypertensive patients who develop de novo albuminuria. Here, we applied multiple proteomic strategy with the purpose to elucidate specific molecular pathways involved in the pathogenesis and provide predictors and chronic organ damage indicators. Briefly, 1143 patients were followed up for a minimum period of 3 years. One hundred and twenty-nine hypertensive patients chronically renin-angiotensin system suppressed were recruited, classified in 3 different groups depending on their albuminuria levels (normoalbuminuria, de novo albuminuria, and sustained albuminuria), and investigated by multiple proteomic strategies. Our strategy allowed us to perform one of the deepest plasma proteomic analysis to date, which has shown 2 proteomic signatures: (1) with predictive value of de novo albuminuria and (2) sustained albuminuria indicator proteins. These proteins are involved in inflammation, immune as well as in the proteasome activation occurring in situations of endoplasmic reticulum stress. Furthermore, these results open the possibility of a future strategy based on anti-immune therapy to treat hypertension which could help to prevent the development of albuminuria and, hence, the progression of kidney damage.N

Valvular Aortic Stenosis: A Proteomic Insight

Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible and effective method to carry out proteomic analysis of stenotic human valves by conventional 2-DE and 2D-DIGE, minimizing the interference due to high calcium concentrations. Furthermore, the protocol permits the aortic stenosis proteome to be analysed, advancing our knowledge in this area

Potential role of new molecular plasma signatures on cardiovascular risk stratification in asymptomatic individuals

The evaluation of cardiovascular (CV) risk is based on equations derived from epidemiological data in individuals beyond the limits of middle age such as the Framingham and SCORE risk assessments. Lifetime Risk calculator (QRisk®), estimates CV risk throughout a subjects' lifetime, allowing those. A more aggressive and earlier intervention to be identified and offered protection from the consequences of CV and renal disease. The search for molecular profiles in young people that allow a correct stratification of CV risk would be of great interest to adopt preventive therapeutic measures in individuals at high CV risk. To improve the selection of subjects susceptible to intervention with aged between 30-50 years, we have employed a multiple proteomic strategy to search for new markers of early CV disease or reported CV events and to evaluate their relationship with Lifetime Risk. Blood samples from 71 patients were classified into 3 groups according to their CV risk (healthy, with CV risk factors and with a previously reported CV event subjects) and they were analyzed using a high through quantitative proteomics approach. This strategy allowed three different proteomic signatures to be defined, two of which were related to CV stratification and the third one involved markers of organ damage.This work was supported by grants from the Instituto de Salud Carlos III (PI070537, IF08/3667-1, PI11-02239, PI 14/01917, PI11/01401, PI11/02432, PI13/01873, PI13/01746, PI13/01581, PI14/01650, PI14/01841), PT13/0001/0013, PIE13/00051, PIE13/00045, CP09/00229, CP15/00129, IDC Salud (3371/002), the MutuaMadrileña Foundation, the SENEFRO Foundation and FONDOS FEDER (RD06/0014/1015, RD12/0042/0071). Sociedad Española de cardiología para la Investigación Básica 2017. Grant PRB3 (IPT17/0019 - ISCIII-SGEFI / ERDF. These results are in line with the Spanish initiative on the Human Proteome Project.S

Urinary exosomes reveal protein signatures in hypertensive patients with albuminuria

Albuminuria is an indicator of cardiovascular risk and renal damage in hypertensive individuals. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control and prevents development of new-onset-albuminuria. A significant number of patients, however, develop albuminuria despite chronic RAS blockade, and the physiopathological mechanisms are underexplored. Urinary exosomes reflect pathological changes taking place in the kidney. The objective of this work was to examine exosomal protein alterations in hypertensive patients with albuminuria in the presence of chronic RAS suppression, to find novel clues underlying its development. Patients were followed-up for three years and were classified as: a) patients with persistent normoalbuminuria; b) patients developing de novo albuminuria; and c) patients with maintained albuminuria. Exosomal protein alterations between groups were identified by isobaric tag quantitation (iTRAQ). Confirmation was approached by target analysis (SRM). In total, 487 proteins were identified with high confidence. Specifically, 48 proteins showed an altered pattern in response to hypertension and/or albuminuria. Out of them, 21 proteins interact together in three main functional clusters: glycosaminoglycan degradation, coagulation and complement system, and oxidative stress. The identified proteins constitute potential targets for drug development and may help to define therapeutic strategies to evade albuminuria progression in hypertensive patients chronically treated.Instituto de Salud Carlos III, fondos FEDER/FSE (PI11/01401, PI13/01873, PI14/01841, IF08/3667-1, PI11-02239, PI 14/0917, PI11/02432, PI13/01746, PI14/01650, PI16/01334, PT13/0001/0013, CP09/00229, CP15/00129, CPII15/00027), Fundacion SENEFRO, Fundacion Conchita Rabago de Jimenez Diaz, and Redes Tematicas de Investigacion Cooperativa (fondos FEDER/FSE, RD12/0021/0001, RD12/0042/0071). These results are lined up with the Spanish initiative on the Human Proteome Project (SpHPP).S