Purification of the c-erbB2/neu membrane-spanning segment:: a hydrophobic challenge

High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography–ESI TOF mass spectrometry.     

Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization

A method is presented for the analysis of peptides in plasma at picomole to femtomole levels. Peptides are isolated from plasma by solid-phase extraction, the peptide of interest is purified by reversed-phase high-performance liquid chromatography (HPLC) and selectively digested using immobilized trypsin or chymotrypsin to yield specific di- or tripeptides. These di- and tripeptides are esterified using heptafluorobutyric anhydride, alkylated with pentafluorobenzyl bromide, then quantified by gas chromatography-mass spectrometry with negative ion chemical ionization. This method has been evaluated for a model synthetic heptapeptide, using a deuterium labeled analog as an internal standard. The half-life of the heptapeptide in human plasma was found to be 2 min. Extraction efficiencies of a tritiated peptide of similar size to the heptapeptide, [³H]DSLET, from plasma using either C₁₈ or strong cation-exchange columns were 85±3 and 70±2%, respectively. Quantitation of fragments from the heptapeptide indicated that the analysis was linear from 1–50 ng of the heptapeptide per ml of plasma. This method was subsequently employed for pharmacokinetic studies of the biologically active peptide Met-enkephalin-Arg-Gly-Leu, where linearity was obtained from 50 to 1000 ng/ml in rat plasma. This method demonstrated negligible side reaction by-products due to autolysis, and has potential for extensive use given the wide availability of gas chromatography-mass spectrometry.     

Separation and detection techniques for peptides and proteins in stability research and bioanalysis

In this paper, a brief overview of the most commonly used methods for the separation and analysis of peptides and proteins in stability and bioanalysis studies is presented. To investigate the physical stability of peptides and proteins, size-exclusion chromatography and electrophoretic separation techniques are being used, apart from several other methods. To determine the chemical stability of these compounds, separation systems are also important, with informative detection modes, such as various spectroscopic detections, electrochemical detection and mass spectrometric detection. For the bioanalysis of peptides, separation is the most important factor, while the detection must be done at the highest possible level of sensitivity.     

Two-dimensional high-resolution electrophoresis of elastin-derived peptides

Degradation of human aortic elastin in vivo yields a restricted number of differentially sized and charged peptides. Elastin-derived peptides (EDP) can be analyzed by two-dimensional electrophoresis after their extraction from human abdominal aortic tissue by 0.2 M sodium chloride. The peptides were separated according to charge by acetic acid-urea-PAGE and then according to molecular mass by SDS-PAGE. The identity of these peptides as EDP was continued by immunoprecipitation and Western blots. The two-dimensional electrophoretic system can resolve desmosine-like cross-linked EDP of the similar molecular configuration but differing in the number of positively charged amino acid residues. The new separation technique of EDP has the capacity to identify defects in desmosine-like cross-links and may be useful in characterizing abberations in elastin structures.     

Matrix-assisted laser desorption–ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures

In theory, peptide mass fingerprinting by matrix assisted laser desorption–ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower μl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.     

Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.     

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Peptides with anticancer use or potential

Summary.  This review is an attempt to illustrate the diversity of peptides reported for a potential or an established use in cancer therapy. With 612 references, this work aims at covering the patents and publications up to year 2000 with many inroads in years 2001–2002. The peptides are classed according to four categories of effective (or plausible) biological mechanisms of action: receptor-interacting compounds; inhibitors of protein-protein interaction; enzymes inhibitors; nucleic acid-interacting compounds. The fifth group is made of the peptides for which no mechanism of action has been found yet. Incidentally this work provides an overview of many of the modern targets of anticancer research. Received March 3, 2002 Accepted October 3, 2002 Published online January 20, 2003 Author's address: Dr. Yves Louis Janin, UMR 176 CNRS-Institut Curie, 26 rue d'Ulm, F-75248 Paris cedex 05, France, E-mail: yves.janin@curie.u-psud.fr     

Identification of substance P metabolites using a combination of reversed-phase high-performance liquid chromatography and capillary electrophoresis

Gradient elution reversed-phase high-performance liquid chromatographic and capillary electrophoretic separations were optimised to separate substance P (SP) and twelve of its fragments. The methods were applied to a study of the in vivo metabolism of substance P in the rat after intrastriatal injection of the peptide (10 nmol). SP and significant amounts of its N-terminal fragments, SP(1-7) and SP(1-4), were detected but no major C-terminal fragments could be identified. At the concentration studied, the metabolism of SP was shown to follow zero order elimination kinetics with a rate of decay of 0.2 nmol/min. As we have shown that SP(1–4) and SP(1–7) can be produced in vivo in the striatum in relatively large amounts, it is conceivable that these fragments contribute to the overall pharmacological pattern of activity of the parent peptide.     

防御素基因工程研究进展

防御素是一类在生物界广泛存在的、富含半胱氨酸、具有微生物和一些恶性细胞抗性的小分子短肽。它具有抗性谱广,作用机理特殊等优点;因此可以用来研制新型的抗生素类药,并在动植物抗病基域工程上发挥重要作用。概述了防御素在基因工程方面的研究进展,并对其应用前景作了展望。     

Cell Penetrating Peptides and Cationic Antibacterial Peptides: TWO SIDES OF THE SAME COIN*

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.     

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.     

New marker of bone resorption: hydroxyproline-containing peptide High-performance liquid chromatographic assay without hydrolysis as an alternative to hydroxyproline determination: a preliminary report

A high-performance liquid chromatographic (HPLC) assay for a urinary hydroxyproline-containing peptide (hydroxyproline peptide, HypP) is described. This peptide represents about 50% of urinary hydroxyproline-containing peptides. Its concentration and total 4-hydroxyproline (Hyp) concentration evaluated in 325 urine samples have been shown to be closely correlated (r = 0.972; y = 0.499x − 1.5), which may indicate that the two markers provide the same information. The HypP assay, similar to Hyp assay, is carried out without hydrolysis of urine samples. After the blocking of primary amino acids by o-phthaldialdehyde (OPA) and derivatization of secondary amino acids by 9-fluorenylmethyl chloroformate (FMOC-Cl), the FMOC derivatives of HypP and 3,4-dehydroproline (internal standard) were separated on a strong anion-exchange column and detected fluorimetrically. HypP concentration was calculated by measurement of peak-area ratios of HypP and the hydroxyproline standard. The HypP/creatinine (mmol/mol) ratio in fasting urine samples from healthy adults was found to be 8.2 (S.D. = 1.6, n = 33) in 27–44-year-old premenopausal women and 6.9 (S.D. = 1.7, n = 21) in 28–49-year-old men.     

Rapid Sequestration and Degradation of Somatostatin Analogues by Isolated Brain Microvessels

Somatostatin (SRIF) is a putative peptide neurotransmitter that may interact with brain capillaries following neurosecretion of the peptide. The present studies investigate the binding and metabolism of SRIF analogues in isolated bovine brain microvessels. 125I-[Tyr1]SRIF was rapidly degraded by capillary aminopeptidase with a half-time of approximately 3 min at 23 degrees C. The microvessel aminopeptidase had a low affinity and high capacity for the peptide, Km = 76 microM and Vmax = 74 nmol min-1 mgp-1. 125I-[Tyr11]SRIF was converted to free iodotyrosine at a much slower rate, presumably by a lower-activity endopeptidase. 125I-[Try11]SRIF was rapidly bound by microvessels, whereas another basic peptide, [Tyr8]bradykinin, or an acidic peptide, CCK8, or a neutral peptide, leucine enkephalin, were bound to a considerably less extent. The binding of 125I-[Tyr11]SRIF to the capillaries was nonsaturable up to a concentration of 1 microgram/ml of unlabeled peptide, and the binding reaction was extremely rapid, reaching equilibrium within 5 s at either 0 degrees C or 37 degrees C. Approximately 20% of the SRIF bound by the microvessels was resistant to acid wash and presumably represented internalized peptide. In addition, the 125I-[Tyr11]SRIF bound rapidly to the endothelial cytoskeleton remaining after a 1% Triton X-100 extraction of the microvessels. The peptide-cytoskeletal binding reaction was nonsaturable up to 1 microgram/ml of unlabeled [Tyr11]SRIF, but it was inhibited by 0.5% polylysine or 0.8 M KCl and was stimulated by 1 mM dithiothreiotol. These studies suggest that brain microvessels rapidly sequester and degrade SRIF analogues and that this may represent one mechanism for rapid inactivation of the neuropeptides subsequent to neurosecretion.     

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein's repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33kDa protein on red blood cell membrane. HABPs' possible function in merozoite invasion of erythrocytes is also discussed.     

Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues

In this paper we compared the antibacterial activity of native microcin J25, a peptide antibiotic, with the activities of two analogues obtained by chemical modifications. In the first analogue, the negative charge of glutamic carboxyl group was specifically blocked with an L-glycine methyl ester and in the second the histidine imidazole ring was carbethoxylated. Both analogues decreased notably its antibiotic activity against Escherichia coli and Salmonella newport, strains sensible to the native microcin J25. The biological activity of the carbethoxylated analogue was completely recovered after treatment with hydroxylamine. The extreme importance of both polar residues could be interpreted as specific structural features indispensable for the peptide transportation into the cell, extrusion outside the cell or alternatively to inhibit the RNA-polymerase.     

Determination of endogenous peptides in the porcine brain: possible construction of peptidome,a fact database for endogenous peptides

Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.