Rüdiger Hess

GCAP‐II: Isolation and characterization of the circulating form of human uroguanylin

The systematic isolation of circulating regulatory peptides which generate cGMP as second messenger resulted in the identification of a novel member of the guanylin family. In the present study we describe the purification and amino acid sequence of a new guanylate cyclase C activating peptide (GCAP‐II). GCAP‐II contains 24 amino acids in the following sequence: FKTLRTIANDDCELCVNVACTGCL. Its molecular mass is 2597.7 Da. The 16 C‐terminal amino acids are identical to uroguanylin from human urine. Native and synthetic GCAP‐II activate GC‐C, the specific guanylate cyclase receptor, of cultured human colon carcinoma (T84) cells. GCAP‐II stimulates chloride secretion in isolated human intestinal mucosa mediated by intracellular cGMP increase. GCAP‐II specific antibodies were used to localize the peptide by immunohistochemistry in entero‐endocrine cells of the colonic mucosa.

Peptide bank generated by large-scale preparation of circulating human peptides.

Human hemofiltrate (HF) is a source for the purification of circulating regulatory peptides. HF is obtained in large quantities during treatment of patients suffering from chronic renal failure. We have developed a large-scale method for separating peptides from amounts up to 10,000 1 HF into 300 fractions in a standardized two-step procedure, employing cation-exchange separation, followed by reversed-phase chromatography. These fractions represent a peptide bank containing bioactive, desalted and lyophilized peptides of blood. Screening for and isolation of regulatory human peptides is simplified by using this peptide bank.

Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics.

Proteomics studies aiming at a detailed analysis of proteins, and peptidomics, aiming at the analysis of the low molecular weight proteome (peptidome) offer a promising approach to discover novel biomarkers valuable for different crucial steps in detection, prevention and treatment of disease. Much emphasis has been given to the analysis of blood, since this source would by far offer the largest number of meaningful biomarker applications. Blood is a complex liquid tissue that comprises cells and extra-cellular fluid. The choice of suitable specimen collection is crucial to minimize artificial occurring processes during specimen collection and preparation (e.g. cell lysis, proteolysis). After specimen collection, sample preparation for peptidomics is carried out by physical methods (filtration, gel-chromatography, precipitation) which allow for separation based on molecular size, with and without immunodepletion of major abundant proteins. Differential Peptide Display (DPD) is an offline-coupled combination of Reversed-Phase-HPLC and MALDI mass spectrometry in combination with in-house developed data display and analysis tools. Identifications of peptides are carried out by additional mass spectrometric methods (e.g. online LC-ESI-MS/MS). In the work presented here, insights into semi-quantitative mass spectrometric profiling of plasma peptides by DPD are given. This includes proper specimen selection (plasma vs. serum), sample preparation, especially peptide extraction, the determination of sensitivity (i.e. by establishing detection limits of exogenously spiked peptides), the reproducibility for individual as well as for all peptides (Coefficient of Variation calculations) and quantification (correlation between signal intensity and concentration). Finally, the implications for clinical peptidomics are discussed.

Expression profiling of breast cancer cells by differential peptide display.

Expression profiling of RNAs or proteins has become a promising means to investigate the heterogeneity of histopathologically defined classes of cancer. Peptides, representing degradation as well as processing products of proteins offer an even closer insight into cell physiology. Peptides are related to the turnover of cellular proteins and are capable to reflect disease-related changes in homoeostasis of the human body. Furthermore, peptides derived from tumor cells are potentially useful markers in the early detection of cancer.In this study, we introduced a method called differential peptide display (DPD) for separating, detecting, and identifying native peptides derived from whole cell extracts. This method is a highly standardized procedure, combining the power of reversed-phase chromatography with mass spectrometry. This technology is suitable to analyze cell lines, various tissue types and human body fluids. Peptide-based profiling of normal human mammary epithelial cells (HMEC) and the breast cancer cell line MCF-7 revealed complex peptide patterns comprising of up to 2300 peptides. Most of these peptides were common to both cell lines whereas about 8% differed in their abundance. Several of the differentially expressed peptides were identified as fragments of known proteins such as intermediate filament proteins, thymosins or Cathepsin D. Comparing cell lines with native tumors, overlapping peptide patterns were found between HMEC and a phylloides tumor (CP) on the one hand and MCF-7 cells and tissue from a invasive ductal carcinoma (DC) on the other hand.

Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas

Estrogen-receptor status provides a major biomarker in breast cancer classification and has an important impact on prognosis and treatment options. The aim of this study was to investigate peptide profiles of invasive breast cancer with positive (n=39) and negative receptor status (n=41). Peptide profiles were generated by ‘Differential Peptide Display’, which is an offline-coupled combination of reversed-phase-HPLC and MALDI mass spectrometry. Mass spectrometric data were correlated with the immunohistochemically determined receptor state. Identification of peptides of interest was carried out by additional mass spectrometric methods (eg MALDI-TOF-TOF-MS-MS). Approximately 3000–7000 signals were detected per sample and thymosin alpha-1, an asparaginyl endopeptidase generated cleavage product of the ubiquitous acidic protein prothymosin-alpha, was found to differentiate the tumor samples according to their receptor status with the highest specificity. The concentration of Thymosin alpha-1 was found to be upregulated (n=37) in estrogen-negative cancer samples and downregulated (n=32) in estrogen-positive breast cancer samples. The expression of the precursor protein (Prothymosin-alpha) has been discussed previously as a prognostic factor in breast cancer. It is involved in the ER signal transduction pathway as an anti-coactivator-inhibitor. From our findings we conclude that Thymosin alpha-1 could serve as a surrogate marker in breast cancers and may indicate ER functionality.

Matrix-assisted laser desorption/ionisation mass spectrometry guided purification of human guanylin from blood ultrafiltrate

The purification of the human peptide hormone guanylin 22-115 from blood ultrafiltrate (hemofiltrate, HF) was achieved using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) as the assay system. Screening a peptide bank generated from 5000 1 HF guanylin 22-115 was detected by its molecular mass when adequate conditions for MALDI-MS analysis were chosen. The sensitivity was even better than of the established biological assay system. In addition, the susceptibility towards solvents and salts is strongly reduced. 1.2 mg of the peptide hormone was purified from 10% of the starting material.

Peptidomics Biomarker Discovery in Mouse Models of Obesity and Type 2 Diabetes

Type 2 diabetes mellitus (T2DM) is caused by the failure of the pancreatic beta-cell to secrete sufficient insulin to compensate a decreased response of peripheral tissues to insulin action. The pathological events causing beta-cell dysfunctions are only poorly understood and early markers that would predict islet function are missing. In contrast to immunoassays, unbiased proteomic technologies provide the opportunity to screen for novel marker protein and peptides of T2DM. An important subset of the proteome, peptides and peptide hormones secreted by the pancreas are deregulated in T2DM. The mass range of peptides and small proteins (1-20 kDa) is only sufficiently targeted by peptidomics, a combination of liquid chromatographic and mass spectrometric (MS) peptide analysis. Here, we describe the application of isotope label-free quantitative peptidomics to display and quantify relevant changes in the level of pancreatic peptides and peptide hormones in a preclinical model of T2DM, the Lep(ob)/Lep(ob) mouse. The amino acid sequence of statistical relevant top candidates was determined by MS/MS fragmentation or Edman degradation. The comparison of lean versus obese mice revealed increased levels of islet-specific peptides that can be divided into the following categories 1) the major islet peptide hormones insulin, amylin and glucagon; 2) proinsulin and C-peptide and 3) novel processing products of secretogranin, glucagon and amylin. Furthermore, we found increased levels of proteins and peptides implicated in zymogen granule maturation (syncollin) and nutritional digestion. In summary, our findings demonstrate that peptidomics is a valid approach to screen for novel peptide biomarkers.

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept.

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.

Detection of low-molecular-mass plasma peptides in the cavernous and systemic blood of healthy men during penile flaccidity and rigidity--an experimental approach using the novel differential peptide display technology.

OBJECTIVES To use Differential Peptide Display (DPD) technology to evaluate the patterns of low-molecular-mass peptides and small proteins in the systemic and cavernous blood taken from healthy adult male volunteers during the penile stages of flaccidity and rigidity. Results from basic research implicate a role of various peptides in the control of mammalian penile erectile tissue. Nevertheless, it is not yet known which particular peptides are essential in the regulation of penile flaccidity, tumescence, rigidity, and detumescence. METHODS Five healthy male subjects were exposed to visual and tactile erotic stimuli to elicit penile erection. Whole blood was simultaneously aspirated from the corpus cavernosum and cubital vein during penile flaccidity and rigidity. Plasma aliquots were subjected to DPD analysis by means of matrix-assisted-laser-desorption-ionization mass mapping and electrospray-ionization quadrupole--time-of-flight mass spectrometry. RESULTS High-resolution two-dimensional peptide mass mapping revealed differences in the systemic and cavernous plasma samples related to penile flaccidity and rigidity. Distinct signals were recognized in the cavernous but not in the systemic plasma obtained during flaccidity. These signals were not registered in the plasma samples obtained from the corpus cavernosum during rigid erection. Although one signal was identified as the blood coagulation-activating peptide XIIIa, the remaining two signals could not be related to any known peptide. These signals may represent unknown local peptidergic factors that might be involved in the regulation of penile flaccidity. CONCLUSIONS Our study demonstrates that DPD is a feasible method for detecting differences in the cavernous and systemic blood in relation to the different functional conditions of the penile erectile tissue. Additional studies using DPD should include the analysis of blood samples taken from the cavernous meshwork of healthy subjects during penile tumescence and detumescence to establish DPD as a valuable tool in contemporary corpus cavernosum basic research.

Peptidomic analysis of blood plasma after in vivo treatment with protease inhibitors--a proof of concept study.

Native peptides can be regarded as surrogate markers for protease activity in biological samples. Analysis of peptides by peptidomics allows to monitor protease activity in vivo and to describe the influence of protease inhibition. To elucidate the potential of peptides as markers for in vivo protease inhibition we analyzed plasma samples from animals treated with either the indirect FXa inhibitor FONDAPARINUX or the dipeptidylpeptidase IV inhibitor AB192. Signals correlating with the treatment were subsequently identified and assessed with respect to protease-dependent consensus cleavage motifs and occurrence of downstream targets. It could be shown that regulated peptides were either substrates, products or downstream targets of the inhibited protease. The results from the present study demonstrate that the in vivo analysis of peptides by peptidomics has the potential to broaden the knowledge of inhibitor related effects in vivo and that this method may pave the way to develop predictive biomarkers.