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Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a “training set” for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a “test set.” The signature panel showed sensitivity of 98% (95% confidence interval, 88.7–99.6) and 83% specificity (95% confidence interval, 51.6–97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment. Urinary proteomics is emerging as a powerful non-invasive tool for diagnosis and monitoring of variety of human diseases. We tested whether signatures of urinary polypeptides can contribute to the existing biomarkers for coronary artery disease (CAD). We examined a total of 359 urine samples from 88 patients with severe CAD and 282 controls. Spot urine was analyzed using capillary electrophoresis on-line coupled to ESI-TOF-MS enabling characterization of more than 1000 polypeptides per sample. In a first step a “training set” for biomarker definition was created. Multiple biomarker patterns clearly distinguished healthy controls from CAD patients, and we extracted 15 peptides that define a characteristic CAD signature panel. In a second step, the ability of the CAD-specific panel to predict the presence of CAD was evaluated in a blinded study using a “test set.” The signature panel showed sensitivity of 98% (95% confidence interval, 88.7–99.6) and 83% specificity (95% confidence interval, 51.6–97.4). Furthermore the peptide pattern significantly changed toward the healthy signature correlating with the level of physical activity after therapeutic intervention. Our results show that urinary proteomics can identify CAD patients with high confidence and might also play a role in monitoring the effects of therapeutic interventions. The workflow is amenable to clinical routine testing suggesting that non-invasive proteomics analysis can become a valuable addition to other biomarkers used in cardiovascular risk assessment. Coronary artery disease (CAD) 1The abbreviations used are: CAD, coronary artery disease; CE, capillary electrophoresis; ETD, electron transfer dissociation; ROC, receiver operating characteristic; AUC, area under the ROC curve; CI, confidence interval; DTA, .dta file; LDL, low density lipoprotein; HDL, high density lipoprotein. 1The abbreviations used are: CAD, coronary artery disease; CE, capillary electrophoresis; ETD, electron transfer dissociation; ROC, receiver operating characteristic; AUC, area under the ROC curve; CI, confidence interval; DTA, .dta file; LDL, low density lipoprotein; HDL, high density lipoprotein. is a leading cause of morbidity and mortality worldwide. The underlying molecular causes are still largely unknown but are likely to involve alterations in gene and protein expression (1McGregor E. Dunn M.J. Proteomics of the heart: unraveling disease.Circ. Res. 2006; 98: 309-321Crossref PubMed Scopus (119) Google Scholar). Despite multiple clinical, electrographic, and biochemical characteristics, there are subgroups of patients who progress to severe, life-threatening CAD without many symptoms and signs (2Fazzini P.F. Prati P.L. Rovelli F. Antoniucci D. Menghini F. Seccareccia F. Menotti A. Epidemiology of silent myocardial ischemia in asymptomatic middle-aged men (the Eccis Project).Am. J. Cardiol. 1993; 72: 1383-1388Abstract Full Text PDF PubMed Scopus (61) Google Scholar). For example, patients with type 2 diabetes and the elderly frequently suffer from silent myocardial infarctions with significantly increased risk of complications (3Scognamiglio R. Negut C. Ramondo A. Tiengo A. Avogaro A. Detection of coronary artery disease in asymptomatic patients with type 2 diabetes mellitus.J. Am. Coll. Cardiol. 2006; 47: 65-71Crossref PubMed Scopus (189) Google Scholar). Early diagnosis of CAD in its presymptomatic stage would allow for better, targeted, and hence more effective primary prevention as compared with current clinical recommendations. Proteomics is increasingly used to examine dynamic changes in protein expression providing new insights into cellular processes. Moreover proteomics analyses have already resulted in the identification of clinically useful biomarkers and can assist in diagnosis and disease staging (1McGregor E. Dunn M.J. Proteomics of the heart: unraveling disease.Circ. Res. 2006; 98: 309-321Crossref PubMed Scopus (119) Google Scholar, 4Hanash S. Disease proteomics.Nature. 2003; 422: 226-232Crossref PubMed Scopus (861) Google Scholar, 5Kolch W. Neususs C. Pelzing M. Mischak H. Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery.Mass Spectrom. Rev. 2005; 24: 959-977Crossref PubMed Scopus (256) Google Scholar). Substances contained in body fluids hold an abundance of information and can be used as a dynamic and concurrent gauge for monitoring the well-being of an organism. Urine presents a rich source of information related to the functioning of many internal organs (5Kolch W. Neususs C. Pelzing M. Mischak H. Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery.Mass Spectrom. Rev. 2005; 24: 959-977Crossref PubMed Scopus (256) Google Scholar, 6Hewitt S.M. Dear J. Star R.A. Discovery of protein biomarkers for renal diseases.J. Am. Soc. Nephrol. 2004; 15: 1677-1689Crossref PubMed Scopus (240) Google Scholar, 7O'Riordan E. Goligorsky M.S. Emerging studies of the urinary proteome: the end of the beginning?.Curr. Opin. Nephrol. Hypertens. 2005; 14: 579-585Crossref PubMed Scopus (35) Google Scholar), and the appearance of certain proteins in the blood stream may result in their appearance in the urine either in the intact form or as peptide fragments. The protein and peptide composition of the urine is determined by the function of the glomerular filtration apparatus, proximal tubular absorption of ultrafiltered proteins, and the capacity of the brush border and lysosomal proteolytic machinery to degrade filtered proteins (8D'Amico G. Bazzi C. Pathophysiology of proteinuria.Kidney Int. 2003; 63: 809-825Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Therefore, detection of one or several proteins or polypeptides may provide a signature of a particular pathological process (7O'Riordan E. Goligorsky M.S. Emerging studies of the urinary proteome: the end of the beginning?.Curr. Opin. Nephrol. Hypertens. 2005; 14: 579-585Crossref PubMed Scopus (35) Google Scholar). We hypothesized that proteomics analysis of urine should yield a panel of biomarker peptides useful as additional tools for the diagnosis and monitoring of CAD. Furthermore we aimed to obtain sequences of biomarkers of the CAD signature panel to gain insight into pathogenetic mechanisms and facilitate comparison with currently used biochemical markers. Capillary electrophoresis on-line coupled to electrospray ionization-time-of-flight mass spectrometry (CE-ESI-TOF MS) seems ideally suited for this purpose because of its non-invasive nature, high resolution, and amenability for future adaptation to clinical laboratory analysis (5Kolch W. Neususs C. Pelzing M. Mischak H. Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery.Mass Spectrom. Rev. 2005; 24: 959-977Crossref PubMed Scopus (256) Google Scholar). We enrolled 88 patients with CAD confirmed by coronary angiography. Patients were recruited at the Western Infirmary, Glasgow, UK. Eighty-two of the 88 patients were reassessed after a further 14 weeks, one patient died, and five patients refused to participate at a follow-up examination. At both assessments blood and midstream spot urine samples were collected. Thirty-two subjects with no history of angina, CAD, or peripheral artery disease who were recruited from a local health club and from surgical wards at Gartnavel General Hospital, Glasgow, UK served as controls. Plasma total cholesterol, low density lipoproteins, high density lipoproteins, triglycerides, high sensitivity C-reactive protein, and serum creatinine were assessed using standard biochemical methods. The modification of diet in renal disease formula was used for the estimation of glomerular filtration rate in study participants (9National Kidney FoundationK/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification.Am. J. Kidney Dis. 2002; 39: S1-S266PubMed Google Scholar). Vascular stiffness was assessed by two methods. First, pulse contour analysis of the diastolic pressure decay was used to estimate large (C1) and small artery compliance (C2; HDI/Pulse Wave CR2000, HDI Inc., Eagan, MN) based on a three-element Windkessel model (10Cohn J.N. Finkelstein S. McVeigh G. Morgan D. LeMay L. Robinson J. Mock J. Noninvasive pulse wave analysis for the early detection of vascular disease.Hypertension. 1995; 26: 503-508Crossref PubMed Scopus (424) Google Scholar). Second, the augmentation index of the central aorta was derived from the radial pulse waveform using a generalized transfer function (SphygmoCor pulse wave analysis system, AtCor Medical, West Ryde, New South Wales, Australia) (11Pauca A.L. O'Rourke M.F. Kon N.D. Prospective of a for pressure from the radial artery pressure PubMed Scopus Google Scholar). 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Discovery and of new protein biomarkers for a 2006; Full Text Full Text PDF PubMed Scopus Google that can be used as Our pattern distinguished the presence and of Furthermore we were also to a dynamic of the pattern in to patients no in pattern patients showed changes toward a biomarker results an proteomics analysis risk proteomics can identify the in is into is because the is dynamic and hence can changes W. Mischak H. The molecular of a proteomics in 2005; PubMed Scopus Google Scholar). also to the effects of therapeutic and assist in Furthermore this also a the kidney and CAD on a molecular and may to chronic kidney disease is one of the for CAD D. risk and renal function in the Int. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Zimmerli et al. (Fri,) studied this question.