Multiparametric detection of autoantibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous disease with respect to disease manifestations, disease progression and treatment response. Therefore, strategies to identify biomarkers that help distinguishing SLE subgroups are a major focus of biomarker research. We reasoned that a multiparametric autoantibody profiling approach combined with data mining tools could be applied to identify SLE patient clusters. We used a bead-based array containing 86 antigens including diverse nuclear and immune defense pathway proteins. Sixty-four autoantibodies were significantly (p < 0.05) increased in SLE (n = 69) compared to healthy controls (HC, n = 59). Using binary cut-off thresholds (95% quantile of HC), hierarchical clustering of SLE patients yields five clusters, which differ qualitatively and in their total number of autoantibodies. In two patient clusters the overall accumulated autoantibody reactivity of all antigens tested was 31% and 48%, respectively. We observed a positive association between the autoantibody signature present in these two patient clusters and the clinical manifestation of glomerulonephritis (GLMN). In addition, groups of autoantibodies directed against distinct intracellular compartments and/or biological motifs characterize the different SLE subgroups. Our findings highlight the relevant potential of multiparametric autoantibody detection and may contribute to a deeper understanding of the clinical and serological diversity of SLE.

Is isolation of comprehensive human plasma peptidomes an achievable quest?

The low molecular weight (LMW; <10kDa)* plasma peptidome has been considered a source of useful diagnostic biomarkers and potentially therapeutic molecules, as it contains many cytokines, peptide hormones, endogenous peptide products and potentially bioactive fragments derived from the parent proteome. The small size of the peptides allows them almost unrestricted vascular and interstitial access, and hence distribution across blood-brain barriers, tumour and other vascular permeability barriers. Therefore, the peptidome may carry specific signatures or fingerprints of an individual's health, wellbeing or disease status. This occurs primarily because of the advantage the peptidome has in being readily accessible in human blood and/or other biofluids. However, the co-expression of highly abundant proteins (>10kDa) and other factors present inherently in human plasma make direct analysis of the blood peptidome one of the most challenging tasks faced in contemporary analytical biochemistry. A comprehensive compendium of extraction and fractionation tools has been collected concerning the isolation and micromanipulation of peptides. However, the search for a reliable, accurate and reproducible single or combinatorial separation process for capturing and analysing the plasma peptidome remains a challenge. This review outlines current techniques used for the separation and detection of plasma peptides and suggests potential avenues for future investigation. This article is part of a Special Issue entitled: HUPO 2014.

Challenges in planning and conducting diagnostic studies with molecular biomarkers.

Biomarkers are of increasing importance for personalized medicine in many areas of application, such as diagnosis, prognosis, or the selection of targeted therapies. In many molecular biomarker studies, intensity values are obtained from large scale ­omics experiments. These intensity values, such as protein concentrations, are often compared between at least two groups of subjects to determine the diagnostic ability of the molecular biomarker. Various prospective or retrospective study designs are available for molecular biomarker studies, and the biomarker used may be univariate or may even consist in a multimarker rule. In this work, several challenges are discussed for the planning and conduct of biomarker studies. The phases of diagnostic biomarker studies are closely related to levels of evidence in diagnosis, and they are therefore discussed upfront. Different study designs for molecular biomarker studies are discussed, and they primarily differ in the way subjects are selected. Using two systematic reviews from the literature, common sources of bias of molecular diagnostic studies are illustrated. The extreme selection of patients and controls and verification bias are specifically discussed. The pre-analytical and technical variability of biomarker measurements is usually expressed in terms of the coefficient of variation, and is of great importance for subsequent validation studies for molecular biomarkers. It is finally shown that the required sample size for biomarker validation quadratically increases with the coefficient of variation, and the effect is illustrated using real data from different laboratory technologies.

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.

Peptidomics: the comprehensive analysis of peptides in complex biological mixtures.

Progress in the sequencing of genomes has resulted in an increasing demand for a functional analysis of gene products in order to understand the underlying physiology. Proteomics has established itself as a highly valuable technology for producing functionally related data in an unparalleled fashion, but is methodologically restricted to the analysis of proteins with higher molecular masses (>10 kDa). The development of a technology which covers peptides with low molecular weight and small proteins (0.5 to 15 kDa) was necessary, since peptides, amongst them families of hormones, cytokines and growth factors, play a central role in many biological processes. To summarise the technologies used for this approach the term "peptidomics" is introduced. In this article, we present the rationale and first results of a novel, universal peptide display approach for the analysis and visualisation of peptides and small proteins from biological samples. Special attention is given to samples derived from extracellular fluids such as blood plasma and cerebrospinal fluid. Additionally, a high throughput identification procedure for the analysis of peptides in their native and processed molecular form is outlined.

Peptidomics technologies for human body fluids.

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.

LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.

Posttranslationally processed forms of the human chemokine HCC-1.

HCC-1 is the only CC-chemokine known so far which circulates in nanomolar concentrations in human plasma. Its physiological function is not well defined. Posttranslational processing of HCC-1 was shown to modulate its biological properties. In this study several different processed forms of HCC-1 were isolated. Western blot analysis of human plasma extracts revealed a HCC-1 immunoreactive double band at 8-10 kDa indicating the presence of two distinct HCC-1 peptides. These peptides were isolated from a peptide library of human blood filtrate and represent predominantly HCC-1 (1-74) and glycosylated HCC-1 (1-74). Glycosylated HCC-1 exhibits a molecular mass of 9621 Da due to O-glycosylation at position 7 (Ser-7) with two N-acetylneuraminic acids and the disaccharide N-acetylgalactosamine galactose. Furthermore N-terminally truncated HCC-1 (3-74) and HCC-1 (4-74) were identified in the peptide library. In hemofiltrate approximately 3% of total HCC-1 represents HCC-1 (3-74) and approximately 1% represents HCC-1 (4-74) whereas the major products are nonglycosylated HCC-1 (1-74) and glycosylated HCC-1 (1-74). Our data imply that HCC-1 (1-74), HCC-1 (3-74), HCC-1 (4-74) and glycosylated HCC-1 (1-74) circulate in human blood. The N-terminal processing and modification of HCC-1 might be of importance in displaying its full biological activity.

Composition of the peptide fraction in human blood plasma: database of circulating human peptides.

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, beta2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aalpha-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate.

We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification.

Equine cardiodilatin/ atrial natriuretic peptide. Primary structure and immunohistochemical localization in auricular cardiocytes.

Cardiodilatin (CDD)/atrial natriuretic peptide (ANP) is a 28-amino acid peptide hormone known to be synthesized in the heart of a large number of different vertebrates. It plays an important role in the regulation of blood pressure and natriuresis/diuresis. Since the cardiovascular system of the horse has to meet the highest requirements concerning its physiological performance, we intended to characterize the cardiodilatin/atrial natriuretic peptide system of this species. By means of immunohistochemistry and immunoelectron microscopy, we precisely identified auricular cardiocytes as the loci of CDD/ANP synthesis. Using aortic smooth muscle relaxation assay and CDD/ANP-ELISA, we succeeded in isolating the biologically active prohormone. We subsequently cloned the equine cDNA of the CDD/ANP precursor protein and deduced its primary sequence. The entire precursor protein is in good agreement with the CDD/ANP prohormones of other mammals. The deduced theoretical average Mr of equine CDD/ANP-1-126 is 13,764, corresponding to the molecular weight of purified peptide determined by ESI-MS. Our findings suggest that equine CDD/ANP is produced in auricular cardiocytes and the predominant storage form of CDD/ANP in the auricle is the prohormone CDD/ANP-1-126.

Human beta-defensin-1: A urinary peptide present in variant molecular forms and its putative functional implication.

Human beta-defensin-1 (hBD-1) was first isolated from blood filtrate by our group. Further studies elucidate the significance of this peptide in the human urogenital tract. The hBD-1 gene is expressed in urogenital epithelial organs such as urinary bladder, ureter, vagina and particularly in distal tubular cells of the kidney. Functional characterization of hBD-1 was carried out with native hBD-1 purified from human body fluids. Several different N-terminally truncated variants derived from the 68-amino acid-containing precursor of hBD-1 occur in blood filtrate and in urine. The generation of these variants can be explained by digestion through a chymotrypsin-like protease. Unlike the alpha-defensins which are structurally related peptide antibiotics, our results indicate that native hBD-1 exhibits minor antimicrobial activity which is not related to the extension of the N-terminus. Only few microorganisms, for example bacilli, are significantly inhibited by hBD-1. Moreover, antibiotic activity is suppressed in solutions containing physiological sodium chloride concentrations. This is in contrast to previous reports assuming a pivotal role of hBD-1 in antimicrobial host defense. In contrast to its weak antimicrobial activity, it is shown that hBD-1 has a strong cytotoxic potential towards mammalian cells like NIH-3T3 fibroblasts. We assume that this property might be important during eradicative processes at epithelia in particular when the synthesis rate of this peptide is upregulated.

HCC-2, a human chemokine: gene structure, expression pattern, and biological activity.

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named "HCC-2." The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKbeta8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1gamma, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1alpha. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.

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.

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.

Structural and functional characterization of vitronectin-derived RGD-containing peptides from human hemofiltrate.

Bioactive peptides derived from the adhesive plasma protein vitronectin are present at submicromolar concentrations in human hemofiltrate of patients with renal diseases and were isolated by a combination of high-efficiency chromatographic steps. The structural and functional properties of these peptides were characterized. Sequencing and mass spectrometry revealed the existence of peptide isoforms (5-6 kDa) which corresponded to the N-terminus (residues 1 to 44-50) of vitronectin. The isolated peptides bound directly to plasminogen-activator inhibitor-1 (PAI-1) and were effective competitors of the interaction of PAI-1 with isolated intact vitronectin or extracellular matrix. These functional properties were indistinguishable from the binding properties of a recombinant fusion protein containing residues 1-52 of vitronectin linked to a portion of glutathione S-transferase, expressed in Escherichia coli. Peptides containing the RGD sequence of vitronectin competed for vitronectin binding to the alpha v beta 3 integrin. No indication for direct growth-factor binding was noted, whereas natural peptides were found associated with PAI-1 as the major binding protein in plasma. These data demonstrate that functionally active vitronectin-derived peptides are released by unknown protease(s) from the mature protein and that these peptides are identical, in terms of activity, to recombinant vitronectin fragments. These natural peptides may interact with active PAI-1 in plasma or at extravascular sites and thereby interfere with established biological functions of intact vitronectin.

Specific determination of tyrosine-phosphorylated proteins and peptides by differential iodination.

A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.

Urodilatin is involved in sodium homeostasis and exerts sodium-state-dependent natriuretic and diuretic effects.

Urodilatin is involved in sodium homeostasis exerts sodium-state-dependent natriuretic and diuretic cts. Eight male volunteers participated in a study consisting of three consecutive phases of 7 days each. The volunteers a sodium diet with 52, 172.6, and 347.8 mmol um/day. Sodium excretion increased from 57.4 +/- 3.7 via .8 +/- 4.6 (P < 0.001) to 322.5 +/- 10.2 mmol/24 h (P < 0.001) at the end of each sodium diet. Urinary urodilatin excretion increased from 24.8 +/- 3.0 via 35.5 +/- 9.0 (P = 0.07) to 49.0 = mol/min (P < 0.01). At the end of each diet, urodilatin was infused for 2 h at 20 ng.kg body wt-1.min-1. Natriuresis increased after low- (4.1 to 52.9 mmol/h, P < 0.001), normal (6.9 to 44.9 mmol/h, P < 0.05), and high-sodium diet (20.1 to 102.9 mmol/h, P < 0.001). Diuresis increased from 174 to 709 (P < 0.001), 395 to 1,026 (P < 0.05), and 266 to 1,339 ml/h < 0.001). The present results indicate that endogenous urodilatin plays an important role in sodium homeostasis and that renal response to exogenous urodilatin is modulated by sodium balance.

Isolation of human uteroglobin from blood filtrate.

The purpose of this study was to assess the possibility of isolating biologically active peptides from human blood using large volumes of blood filtrate, which are available from patients undergoing extracorporeal ultrafiltration because of renal insufficiency. This filtrate was submitted to six chromatographic separation steps, yielding one purified peptide which was completely analysed in its primary structure. It was found to be strikingly similar to proteins, described initially as rabbit uteroglobin (or blastokinin) and, more recently, from human bronchial lavage as the '10 kDa Clare cell protein', as well as from human urine as 'protein-1'. The natural molecule contains two chains of identical amino acid sequences of 70 residues which are arranged as an antiparallel dimer due to the disulphide bonds between two cysteines at positions 3 and 69. Mass analysis of the molecular forms yielded molecular weights from 15827 Da (non-oxidized form) to 15859 Da (bi-oxidized form). We conclude that this peptide isolated from the filtrate represents the human uteroglobin, and we demonstrate for the first time that this peptide may be involved as a humoral factor in reproductive or other physiological functions.

[Urodilatin (INN: ularitide). A new peptide in the treatment of acute kidney failure following liver transplantation]. / Urodilatin (INN: Ularitide). Ein neues Peptid in der Intensivbehandlung des akuten Nierenversagens nach Lebertransplantation.

UNLABELLED: Acute renal failure (ARF) is a serious complication following liver transplantation. Many therapeutic regimens have been used so far but with limited success. Urodilatin (URO) is a new member of the atrial natriuretic peptide (ANP) family. When administered intravenously, URO induces strong diuresis and natriuresis with tolerable hemodynamic side effects. Preliminary non-controlled clinical studies demonstrate beneficial effects using URO as a therapeutic agent in patients suffering from ARF following heart and liver transplantation (HTx, LTx). These results prompted us to initiate this first controlled clinical trial to investigate whether URO infusion can improve renal function in patients with emerging ARF following LTx. METHOD: We initiated a randomized, double-blind, placebo-controlled study comparing five patients receiving i.v. URO infusion (20 ng/kg bw/min) with four placebo patients after informed consent was obtained. Optional inclusion criteria were oliguria/anuria ( < 0.5 ml/kg/h), refractory to conventional treatment including administration of furosemide and dopamine, increase of serum creatinine to a least 200% of preoperative values, and BUN levels > or = 25 mmol/l. The primary parameters for efficacy was the frequency of hemodialysis/hemofiltration. RESULTS: The frequency of hemodialysis/hemofiltration during URO or placebo infusion was significantly reduced (P = 0.03) in the URO-treated patients in comparison with placebo. BUN levels did not differ between two groups, but serum creatinine levels were consistently lower in the URO group. Diuresis tended to be stronger in the URO group, maintaining high levels despite a significant reduction in the administration of furosemide in comparison with placebo. CONCLUSION: We conclude that URO seems to be a new approach for the treatment of therapy-resistant postoperative ARF following LTx.