Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease.

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Comparison of the amniotic fluid and fetal urine peptidome for biomarker discovery in renal developmental disease.

Production of amniotic fluid (AF) is view as predominately driven by excretion of fetal urine (FU). However, the origin of AF peptides, often considered as potential biomarkers of developmental diseases, has never been investigated. Here, we evaluated the FU origin of AF peptides and if the AF peptide content can be used as a surrogate of FU. The abundance of endogenous peptides was analyzed by capillary electrophoresis coupled to mass spectrometry in 216 AF and 64 FU samples. A total of 2668 and 3257 peptides was found in AF and FU respectively. The AF peptidome largely overlapped with the FU peptidome, ranging from 54% in the second pregnancy trimester to 65% in the third trimester. Examination of a subset of 16 paired AF and FU samples revealed that 67 peptides displayed a significant positively correlated abundance in AF and FU, strongly suggesting that their presence in AF was directly associated to FU excretion. As proof-of-concept we showed that measuring the AF abundance of these 67 peptides of FU origin allowed prediction of postnatal renal survival in fetuses with posterior urethral valves. These results demonstrate that the AF peptidome can be considered as a good surrogate of the FU peptidome.

Capillary electrophoresis coupled to mass spectrometry to establish polypeptide patterns in dialysis fluids.

Combination of capillary electrophoresis with mass spectrometry (CE-MS) allows generation of polypeptide patterns of body fluids. In a single CE-MS (45 min) run more than 600 polypeptides were analyzed in hemodialysis fluids obtained with different membranes (high-flux/low-flux). Larger polypeptides (M(r) > 10 000) were almost exclusively present in high-flux dialysates only, while in low-flux dialysates additional small polypeptides were detected. Comparison to the normal urine pattern yielded a surprisingly low consensus: a number of polypeptides present in urine were missing. We established a fast and sensitive technique, easily applicable to the monitoring of different modalities of dialyzers.

Determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers.

The on-line coupling of capillary electrophoresis (CE) with electrospray-time-of-flight mass spectrometry (MS) has been used to obtain patterns of peptides and proteins present in the urine of healthy human individuals. This led to the establishment of a "normal urine polypeptide pattern", consisting of 247 polypeptides, each of which was found in more than 50% of healthy individuals. Applying CE-MS to the analysis of urine of patients with kidney disease revealed differences in polypeptide pattern. Twenty-seven polypeptides were exclusively found in samples of patients. Another 13, present in controls, were missing. These data indicate that CE-MS can be applied as powerful tool in clinical diagnostics.

Human urinary peptide database for multiple disease biomarker discovery.

PURPOSE: Human urine is an ideal candidate for use in clinical diagnostics. It is easily available, as untrained personnel can collect it. It correlates well with the pathophysiology of a number of diseases, making it a useful source for clinical proteomics. EXPERIMENTAL DESIGN: In this article, we give an update of the human urinary peptide database derived from over 13,000 data sets of CE-MS by now. RESULTS: Urine samples from both patients and healthy subjects were analyzed by CE-MS; these included 47 different pathophysiological conditions. Besides defining biomarkers by their experimental parameters, information on their sequences provides fundamental data into the pathological pathways of diseases. Therefore, we have sequenced 953 urinary peptides by using state-of-the-art top-down MS/MS. Identified biomarkers of all clinical proteomic CE-MS studies including their regulation are also listed in this work. CONCLUSIONS AND CLINICAL RELEVANCE: Biomarker discovery can be used in the management of a wide range of diseases, by combining these data sets of the database. Taking this approach, we can reveal details, at a molecular level, on the pathogenesis of a number of diseases, in particular those associated with urine production and excretion.

CE-MS analysis of the human urinary proteome for biomarker discovery and disease diagnostics.

Owing to its availability, ease of collection, and correlation with pathophysiology of diseases, urine is an attractive source for clinical proteomics. However, many proteomic studies have had only limited clinical impact, due to factors such as modest numbers of subjects, absence of disease controls, small numbers of defined biomarkers, and diversity of analytical platforms. Therefore, it is difficult to merge biomarkers from different studies into a broadly applicable human urinary proteome database. Ideally, the methodology for defining the biomarkers should combine a reasonable analysis time with high resolution, thereby enabling the profiling of adequate samples and recognition of sufficient features to yield robust diagnostic panels. Capillary electrophoresis coupled to mass spectrometry (CE-MS), which was used to analyze urine samples from healthy subjects and patients with various diseases, is a suitable approach for this task. The database of these datasets compiled from the urinary peptides enabled the diagnosis, classification, and monitoring of a wide range of diseases. CE-MS exhibits excellent performance for biomarker discovery and allows subsequent biomarker sequencing independent of the separation platform. This approach may elucidate the pathogenesis of many diseases, and better define especially renal and urological disorders at the molecular level.

Proteomic patterns established with capillary electrophoresis and mass spectrometry for diagnostic purposes.

BACKGROUND: Proteomics applied in large scale may provide a useful diagnostic tool. METHODS: We developed an online combination of capillary electrophoresis with mass spectrometry, allowing fast and sensitive evaluation of polypeptides found in body fluids. Utilizing this technology, polypeptide patterns from urine are established within 45 minutes. About 900 to 2500 polypeptides as well as their concentrations are detected in individual urine samples without the need for specific reagents such as antibodies. To test this method for clinical application, we examined spot urine samples from 57 healthy individuals, 16 patients with minimal change disease (MCD), 18 patients with membranous glomerulonephritis (MGN), and 10 patients with focal segmental glomerulosclerosis (FSGS). RESULTS: One-hundred seventy-three polypeptides were present in more than 90% of the urine samples obtained from healthy individuals, while 690 polypeptides were present with more than 50% probability. These data permitted the establishment of a "normal" polypeptide pattern in healthy individuals. Polypeptides found in the urine of patients differed significantly from the normal controls. These differences allowed the distinction of specific protein spectra in patients with different primary renal diseases. Abnormal pattern of proteins were found even in urine from patients in clinical remission. CONCLUSION: The data indicate that capillary electrophoresis with mass spectrometry coupling provides a promising tool that permits fast and accurate identification and differentiation of protein patterns in body fluids of healthy and diseased individuals, thus enabling diagnosis based on these patterns.