Towards characterization of the human urinary peptidome.

Biomarker discovery in human urine has become an evolving and potentially valuable topic in relation to renal function and diseases of the urinary tract. In order to deliver on the promises and to facilitate the development of validated biomarkers or biomarker panels, protein and peptide profiling techniques need high sample throughput, speed of analysis, and reproducibility of results. Here, we outline the performance characteristics of the liquid chromatography/MALDI-TOF-MS based differential peptide display (DPD(1)) approach for separating, detecting, abundance profiling and identification of native peptides derived from human urine. The typical complexity of peptides in human urine (resolution of the technique with respect to detectable number of peptides), the reproducibility (coefficient of variation for abundance profiles of all peptides detected in biological samples) and dynamic range of the technique as well as the lower limit of detection were characterized. A substantial number of peptides present in normal human urine were identified and compared to findings in four published proteome studies. In an explorative approach, pathological urines from patients suffering from post-renal-filtration diseases were qualitatively compared to normal urine. In conclusion, the peptidomics technology as shown here has a great potential for high throughput and high resolution urine peptide profiling analyses. It is a promising tool to study not only renal physiology and pathophysiology and to determine new biomarkers of renal diseases; it also has the potential to study remotely localized or systemic aberrations within human biology.

Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry.

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.

Peptidomic approaches in proteomic research.

Peptides play a central role in many physiological processes. In order to comprehensively analyze all peptides and small proteins of a whole organism or a subsystem (peptidome), technologies other than 2-D gel electrophoresis are required. Although systematic efforts directed at peptides and peptidomes, comparable to the numerous proteomics projects, are largely lacking to date, a number that employ liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of a variety of different peptide sources. Furthermore, distinct peptide classes, such as antimicrobial peptides or peptides related to metabolic diseases such as diabetes/obesity, are once again receiving attention. Here we discuss peptides in terms of their applicability to serve as diagnostic markers (or more generally as biomarkers), as well as therapeutic targets or lead compounds. There are also a number of technological challenges that need to be overcome in the study of potent animal venoms and plant toxins, both of which are generally peptides and which are discussed as potential lead compounds for therapeutic intervention in diseases such as cancer.

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases.

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.

In vivo profiling of DPP4 inhibitors reveals alterations in collagen metabolism and accumulation of an amyloid peptide in rat plasma.

Dipeptidyl peptidase 4 (DPP4) inhibitors represent a novel class of oral anti-hyperglycemic agents. The complete pharmacological profile of these protease inhibitors remains unclear. In order to gain deeper insight into the in vivo effects caused by DPP4 inhibition, two different DPP4 inhibitors (vildagliptin and AB192) were analyzed using differential peptide display. Wistar rats were treated with the DPP4 inhibitors (0.3mgkg(-1); 1mgkg(-1) or 3mgkg(-1) body weight) and DPP4 activity was measured before and at the end of the experiment. One hour after compound administration, blood plasma samples were collected to generate peptide displays and to subsequently identify differentially regulated peptides. A dose-dependent decrease in blood plasma DPP4 activity was measured for both inhibitors. DPP4 inhibition influenced collagen metabolism leading to depletion of collagen derived peptides (e.g. collagen alpha 1 (III) 521-554) and accumulation of related N-terminally extended collagen derived peptides (e.g. collagen alpha 1 (III) 519-554). Furthermore, the intact amyloid rat BRI (1-23) peptide was detected in plasma following in vivo DPP4 inhibition. DPP4 catalyzed cleavage kinetics of the BRI peptide were determined in vitro. The k(cat) and K(m) for cleavage by DPP4 were 5.2s(-1) and 14microM, respectively, resulting in a specificity constant k(cat)/K(m) of 0.36 x 10(6)s(-1)M(-1). Our results demonstrate that differential peptide analysis can be applied to monitor action of DPP4 inhibition in blood plasma. For the first time effects on basal collagen metabolism following DPP4 inhibition in vivo were demonstrated and the BRI amyloid peptide was identified as a novel DPP4 substrate.

Dual-radionuclide simultaneous biliary and gastric scintigraphy to depict surgical treatment of bile reflux.

Biliary diversion procedures are performed during gastric surgery to decrease bile reflux. A 1-day dual-radionuclide examination was studied to determine its potential in the evaluation of the effectiveness of the Braun enteroenterostomy in reducing bile reflux and its effects on gastric emptying. Orally ingested gallium 67-labeled egg and intravenously administered technetium 99m diisopropyl-imino-diacetic acid were imaged simultaneously. This provided a way to depict both bile reflux and gastric emptying on the same day in patients who underwent gastric surgery. Overall, the Braun enteroenterostomy trades bile reflux, a symptomatic and premalignant disease, for gastroparesis, a less severe and often treatable disease.