Michael Schrader

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.

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.

Isolation and characterization of the circulating form of human endostatin.

Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10–20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C‐terminal fragment of collagen α1(XVIII) was guided by MALDI‐MS and the exact molecular mass determined by ESI‐MS was found to be 18 494 Da. N‐terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1–3 and 2–4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no anti‐proliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.

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.

The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans

The disulphide bond pattern of the long disintegrin bitistatin (83 amino acids, 14 cysteines) was established using structural information gathered by amino acid analysis, N‐terminal sequencing, and molecular mass determination of fragments isolated by reversed‐phase HPLC after polypeptide degradation with trypsin and oxalic acid. A computer program was used to calculate all possible combinations of disulphide‐bonded peptides matching the mass spectrometric data, and the output was filtered using compositional and sequence data. Disulphide bonds between cysteines 16–34, 18–29, 28–51, 42–48, 47–72, and 60–79 are conserved in medium‐long disintegrins flavoridin and kistrin (70 amino acids, 12 cysteines), and the two cysteine residues at positions 5 and 24 found in bitistatin but not in other disintegrin molecules are disulphide‐bridged. This linkage creates an extra, large loop, which, depending on whether the NMR structure of flavoridin or kistrin is used for modelling the structure of bitistatin, lies opposite or nearly parallel, respectively, to the biologically active RGD‐containing loop.

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.

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.

DISTINCTION BETWEEN THE THREE DISULFIDE ISOMERS OF GUANYLIN 99-115 BY LOW-ENERGY COLLISION-INDUCED DISSOCIATION

Guanylin 99-115 is a small human peptide hormone with two disulfide bonds. The four cysteinyl residues in this peptide allow the formation of two disulfide bridges in three different ways but only the 1-3/2-4 combination is able to bind to the receptor and cause an increase of intracellular cGMP, while the other isomers are biologically inactive. Using guanylin 99-115 as a model peptide, the aim of this study was to investigate whether it is possible to distinguish the differently bridged isomers directly by tandem mass spectrometry. Guanylin isomers were generated by performing an air oxidation of fully reduced guanylin 1-3/2-4 obtained by chemical synthesis. The reaction product is a mixture of guanylin 1-4/2-3 and guanylin 1-2/3-4 in a ratio of 3:1, but there is virtually no guanylin 1-3/2-4. The two biologically inactive peptides were separated by reversed-phase high pressure liquid chromatography (HPLC). Using low-energy collision-induced dissociation tandem mass spectrometry, it was possible to distinguish unambiguously between the three guanylin isomers. This was possible due to the identification of a large number of fragments with intact disulfide bonds. Accordingly, this strategy of a direct and sensitive analysis should work as well for other peptides, with the potential to determine an undefined disulfide bond pattern.