Elucidation of retention mechanism of dipeptides on a ristocetin A-based chiral stationary phase using a combination of chromatographic and molecular simulation techniques.

Two chiral stationary phases virtually reproducing the Nautilus-R column were modeled in silico to study the enantiorecognition mechanism of some selected dipeptides, taking into consideration the two different anchoring alternatives to the silica layer involving the two ristocetin A amino groups. A mobile phase composed of water-methanol (40:60, v/v) was included in the system. The analyses of the trajectories supported the experimental L(LL)

Enantioselective adsorption dynamics of leucyl-leucine in a Chirobiotic R column.

The dynamics of adsorption of the Leu-Leu stereoisomers in a chromatographic column packed with the Chirobiotic R chiral stationary phase bearing grafted antibiotic ristocetin A was studied by means of measurement and analysis of van Deemter plots. Similar measurements were carried out with weakly retained Gly-Gly for the sake of comparison. The bulk diffusion coefficients of the investigated dipeptides were also determined. It is found that the van Deemter plots of both the Leu-Leu stereoisomers and Gly-Gly have an uncommon convex-upward shape. Besides, the van Deemter B coefficients for the Leu-Leu stereoisomers, but not for Gly-Gly, have unusually high values. It is suggested that a high transcolumn contribution to eddy dispersion, which turned out to be enantioselective, accounts for these findings. Adsorption kinetics of all the dipeptides considered is relatively slow, the adsorption rate constant (kads) being of order of magnitude 20-60 s-1. kads does not depend on the configuration of Leu-Leu stereoisomers, although their affinity toward the chiral selector depends on this factor. This supports the above hypothesis that eddy dispersion is mainly responsible for the observed peculiarities in the dynamic behavior of dipeptides, and adsorption kinetics has secondary importance in this phenomenon.

Enantioselective retention mechanisms of dipeptides on antibiotic-based chiral stationary phases: Leucyl-leucine, glycyl-leucine, and leucyl-glycine as case studies.

Chromatographic behaviors of dipeptides consisting of leucine and glycine were studied on two antibiotic-based chiral stationary phases (CSPs) with teicoplanin (Chirobiotic T) or ristocetin A (Chirobiotic R) as chiral selectors under reversed-phase conditions. The effect of mobile phase pH on the retention of stereoisomers of dipeptides was investigated and thermodynamic characteristic of adsorption were measured at different pH values. It was shown that the retention of dipeptides depends on the ionization of their molecules in the mobile phase, as different ionic forms have different affinity towards antibiotic selectors. Enantioselectivity of the bound antibiotics with respect to Leu-Leu stereoisomers was achieved via steric modulation of ion-ion interactions between the solute and the selector, while in the case of Gly-Leu enantiomers non-ionic interactions such as hydrogen bonding might play the key role. In both cases, the dipeptides terminating in D-Leu were retained stronger than their optical antipodes, whereas the enantiomers of Leu-Gly were hardly separated. The regression analysis of the retention data applying the Horvath-Melander-Molnar model revealed that different types of enantioselectivity resides in particular ionic forms of the compounds: cations are responsible for the separation of diastereomeric pairs and the anionic and zwitterionic forms have a universal enantioselectivity on the Chirobiotic T CSP, and the anions and zwitterions are the enantioselective forms for the Chirobiotic R CSP.

Unusual van Deemter plots of optical isomers on a chiral brush-type liquid chromatography column.

Unusual dynamic behavior of the enantiomers of 9-bromo-11b-(tert-butyl)-2,3,6,11b-tetrahydrooxazolo[3',2':1,5]pyrrolo[3,4-b]quinoline-5,11-dione (I) was observed on a Nautilus-R column packed with silica grafted with antibiotic ristocetin. It consisted in (i) antibatic behavior of the van Deemter plots of the enantiomers and (ii) high and strongly enantiomer dependent values of the A- and B-terms of the van Deemter equation. Although rare, such a pattern has been found earlier in chiral chromatography, with all reported cases limited to brush-type chiral stationary phases. Adsorption dynamics in this system was studied by means of the moment method and the peak parking technique; hydrodynamic properties of the column were explored by using unretained tracers. It was shown that the peculiar shape of the van Deemter curves for the enantiomers of I is conditioned by imperfect packing of the stationary phase, which result in high transcolumn eddy dispersion, and by slow adsorption/desorption kinetics. It was proven that the whole void volume of the column available to an eluent is not accessible to the studied analyte because it cannot penetrate the space between neighboring grafted ligands. Its mass transfer in pores is also affected by the fact that the stagnant layer of the binary water-acetonitrile mobile phase differs in composition from the bulk liquid due to preferential adsorption of water that influences the apparent molecular diffusivity of solutes. An effect of the structures of analyte and chiral selector on the adsorption kinetics is also briefly discussed.

Trichlorination of a Teicoplanin-Type Glycopeptide Antibiotic by the Halogenase StaI Evades Resistance.

Glycopeptide antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram-positive pathogens. These antibiotics are specialized metabolites produced by several genera of actinomycete bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68-kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all necessary genes for the synthesis of A47934, including two predicted halogenase genes, staI and staK In this study, we report that only one of the halogenase genes, staI, is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for antibiotic activity, as assessed by binding affinity for the target N-acyl-d-Ala-d-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of the enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of antibiotic.

The physical spacing between the von Willebrand factor D'D3 and A1 domains regulates platelet adhesion in vitro and in vivo.

Essentials The role of von Willebrand factor (VWF) domains in regulating platelet adhesion was studied in vivo. Multimeric VWF with spacers at the N- and C-terminus of VWF-A1 were systematically tested. N-terminal modified VWF avidly bound platelet GpIbα, causing VWD Type2B like phenotype in mice. Novel anti-D'D3 mAbs suggest that changes at the D'D3-A1 interface may be biologically relevant. SUMMARY: Background Previous ex vivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF in vivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6 nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested ex vivo and in vivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF-/- mice caused profound thrombocytopenia, reduced plasma VWF concentrations, lower multimer distribution, and incessant tail bleeding that is reminiscent of von Willebrand disease type 2B. Platelet plugs were noted in the portal veins and hepatic arteries. An anti-D'D3 mAb DD3.3 that displays enhanced binding to VWF containing the N-terminal mucin insert also exhibited increased binding to wild-type VWF under shear and upon ristocetin addition. Conclusion Conformation changes at the VWF D'D3-A1 interface may be a key regulator of thrombosis in vivo. Structural features at the A1-A2 interface are likely of less significance.

More than just recruitment: the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH.

Glycopeptide antibiotic biosynthesis involves a complex cascade of reactions centred on a non-ribosomal peptide synthetase and modifiying proteins acting in trans, such as Cytochrome P450 enzymes. These P450s are responsible for cyclisation of the peptide via cross-linking aromatic amino acid side chains, which are a hallmark of the glycopeptide antibiotics. Here, we analysed the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934. Our results demonstrate that the P450 StaH is recruited to the NRPS machinery through interaction with the X-domain present in the last A47934 NRPS module. We determined the crystal structure of StaH and showed that it is responsible for the first cyclisation in A47934 biosynthesis and additionally exhibits flexible substrate specificity. Our results further point out that the X-domain has an impact on the efficiency of the in vitro cyclisation reaction: hybrid PCP-X constructs obtained by domain exchange between A47934 and teicoplanin biosynthesis NRPS modules reveal that the X-domain from A47934 leads to decreased P450 activity and alternate stereochemical preference for the substrate peptide. We determined that a tight interaction between StaH and the A47934 X-domain correlates with decreased in vitro P450 activity: this highlights the need for glycopeptide antibiotic cyclisation to be a dynamic system, with an overly tight interaction interfering with substrate turnover in vitro.

A few atoms make the difference: synthetic, CD, NMR and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycon derivatives.

Despite the close structural similarity between the heptapeptide cores of the glycopeptide antibiotics teicoplanin and ristocetin, synthetically modified derivatives of their aglycons show significantly different antibacterial and antiviral properties. The teicoplanin aglycon derivatives with one exception proved to be potent antibacterials but they did not exhibit anti-influenza virus activity. In contrast, the aglycoristocetin derivatives generally showed high anti-influenza virus activity and possessed moderate antibacterial activity. A systematic structure-activity relationship study has been carried out on ristocetin and teicoplanin aglycon derivatives, to explore which structural differences are responsible for these markedly different biological activities. According to electronic circular dichroism and in silico conformational studies, it was found that the differences in anti-influenza virus activity are mainly determined by the conformation of the heptapeptide core of the antibiotics controlled by the presence or absence of chloro substituents. Knowledge of the bioactive conformation will help to design new analogs with improved anti-influenza virus activity. For the teicoplanin derivatives, it was shown that derivatization to improve the antiviral efficacy was accompanied by a significant decrease in antibacterial activity.

Extracorporeal membrane oxygenation induces short-term loss of high-molecular-weight von Willebrand factor multimers.

BACKGROUND: High-molecular-weight (HMW) von Willebrand factor (vWF) multimers are crucial for primary hemostasis. Increased shear stress from ventricular assist devices can provoke premature degradation of HMW vWF multimers. Whether similar loss of vWF multimers occurs during extracorporeal membrane oxygenation (ECMO) is not clear. METHODS: We conducted a prospective observational study in a clinical cohort of patients who required ECMO for intractable cardiac and/or respiratory failure. The primary end point was the quantity and quality of HMW vWF multimer bands before, during, and after ECMO support. To investigate further changes in primary hemostasis, we also measured vWF antigen activity (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCo), and factor VIII in 38 patients who required ECMO support before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO therapy. RESULTS: Compared with baseline, vWF:Ag and vWF:RCo decreased after 24 hours of ECMO (mean ± SD, vWF:Ag, 307% ± 152% to 261% ± 138%, P = 0.002; vWF:RCo 282% ± 145% to 157% ± 103%, P < 0.0001) and remained lower during ongoing support (vWF:Ag 265% ± 128%, P = 0.025; vWF:RCo 163% ± 94%, P < 0.0001). After termination of ECMO, vWF:Ag was greater than baseline (359% ± 131%, P = 0.004) and vWF:RCo was similar to baseline levels (338% ± 142%, P = 0.046). Compared with baseline, the calculated vWF:RCo/vWF:Ag ratio decreased after 24 hours on support (0.96 ± 0.23 to 0.61 ± 0.17, P ≤ 0.0001) and remained lower during 48 hours on ECMO (0.63 ± 0.18, P ≤ 0.0001). After termination of ECMO support (0.94 ± 0.19, P = 0.437), values rapidly returned to baseline. The number of HMW vWF multimers (n) decreased from baseline after 24 hours on ECMO (21 ± 1.4 to 14 ± 1.8, P ≤ 0.0001) and after 48 hours on ECMO (15 ± 2.1, P ≤ 0.0001). Twenty-four hours after termination of ECMO support, HMW vWF multimeric pattern had returned to baseline values (21 ± 1.8, P = 0.551). CONCLUSIONS: Loss of HMW vWF multimer bands occurred in patients undergoing ECMO support and resolved after the termination of ECMO. Although not detectable with coagulation screening tests, a vWF:RCo/vWF:Ag ratio <0.7 during ECMO was highly indicative for loss of HMW vWF multimers. Our findings may at least in part explain increased bleeding tendency during ECMO therapy. Administration of vWF concentrates may support restoration of primary hemostasis in patients with relevant bleeding during ECMO support.

Performance evaluation and multicentre study of a von Willebrand factor activity assay based on GPIb binding in the absence of ristocetin.

The functional activity of von Willebrand factor (VWF) is most frequently measured by using the ristocetin cofactor assay (VWF:RCo). However, the method's drawbacks include unsatisfactory precision, sensitivity and availability of automated system applications. We have developed an alternative assay (INNOVANCE VWF Ac) that is based on the binding of VWF to recombinant glycoprotein Ib (GPIb). Two gain-of-function mutations were introduced into a GPIb fragment, allowing an assay format without ristocetin. Fully automated assay applications are available for the BCS/BCS XP systems and the Sysmex CS-2000i, Sysmex CA-7000, Sysmex CA-1500 and Sysmex CA-560 systems.The INNOVANCE VWF Ac assay measuring range extends from 4 to 600% VWF for all systems except the Sysmex CA-560 system. Within-device precision values were found to be between 2 and 7%. The limit of detection was below 2.2% VWF. In a study on the BCS XP system, a total number of 580 sample results yielded a correlation to the VWF:RCo assay of r equal to 0.99 (slope = 0.96). Very similar results were observed when von Willebrand disease samples type 1, 2A, 2B, 2M, 2N and 3 were investigated with the new assay and the VWF:RCo assay. The excellent performance data and comparability to VWF:RCo, together with the ease of use, led us to the conclusion that the ristocetin cofactor assay can be replaced by the new GPIb-binding assay to reliably diagnosing patients with von Willebrand disease.

Anti-cooperative ligand binding and dimerisation in the glycopeptide antibiotic dalbavancin.

Dalbavancin, a semi-synthetic glycopeptide with enhanced antibiotic activity compared to vancomycin and teicoplanin, binds to the C-terminal lysyl-d-alanyl-d-alanine subunit of Lipid II, inhibiting peptidoglycan biosynthesis. In this study, micro-calorimetry and electrospray ionization (ESI)-MS have been used to investigate the relationship between oligomerisation of dalbavancin and binding of a Lipid II peptide mimic, diacetyl-Lys-d-Ala-d-Ala (Ac2-Kaa). Dalbavancin dimerised strongly in an anti-cooperative manner with ligand-binding, as was the case for ristocetin A, but not for vancomycin and teicoplanin. Dalbavancin and ristocetin A both adopt an 'closed' conformation upon ligand binding, suggesting anti-cooperative dimerisation with ligand-binding may be a general feature of dalbavancin/ristocetin A-like glycopeptides. Understanding these effects may provide insight into design of novel dalbavancin derivatives with cooperative ligand-binding and dimerisation characteristics that could enhance antibiotic activity.

A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets.

There is substantial clinical interest in synthetic platelet analogs for potential application in transfusion medicine. To this end, our research is focused on self-assembled peptide-lipid nanoconstructs that can undergo injury site-selective adhesion and subsequently promote site-directed active platelet aggregation, thus mimicking platelet's primary hemostatic actions. For injury site-selective adhesion, we have utilized a coagulation factor FVIII-derived VWF-binding peptide (VBP). FVIII binds to VWF's D'-D3 domain while natural platelet GPIbα binds to VWF's A1 domain. Therefore, we hypothesized that the VBP-decorated nanoconstructs will adhere to VWF without mutual competition with natural platelets. We further hypothesized that the adherent VBP-decorated constructs can enhance platelet aggregation when co-decorated with a fibrinogen-mimetic peptide (FMP). To test these hypotheses, we used glycocalicin to selectively block VWF's A1 domain and, using fluorescence microscopy, studied the binding of fluorescently labeled VBP-decorated nanoconstructs versus platelets to ristocetin-treated VWF. Subsequently, we co-decorated the nanoconstructs with VBP and FMP and incubated them with human platelets to study construct-mediated enhancement of platelet aggregation. Decoration with VBP resulted in substantial construct adhesion to ristocetin-treated VWF even if the A1-domain was blocked by glycocalicin. In comparison, such A1-blocking resulted in significant reduction of platelet adhesion. Without A1-blocking, the VBP-decorated constructs and natural platelets could adhere to VWF concomitantly. Furthermore, the constructs co-decorated with VBP and FMP enhanced active platelet aggregation. The results indicate significant promise in utilizing the FVIII-derived VBP in developing synthetic platelet analogs that do not interfere with VWF-binding of natural platelets but allow site-directed enhancement of platelet aggregation when combined with FMP.

Enantiomeric separation of underivatized amino acids: predictability of chiral recognition on ristocetin a chiral stationary phase.

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high-performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R.

Comparison of a new chemiluminescent immunoassay for von Willebrand factor activity with the ristocetin cofactor-induced platelet agglutination method.

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50 IU dL(-1) ): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50µM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold.

Although glycopeptide antibiotics (GPAs), including vancomycin and teicoplanin, represent the most important class of anti-infective agents in the treatment of serious gram-positive bacterial infections, their usefulness is threatened by the emergence of resistant strains. GPAs are complex natural products consisting of a heptapeptide skeleton assembled via nonribosomal peptide synthesis and constrained through multiple crosslinks, with diversity resulting from enzymatic modifications by a variety of tailoring enzymes, which can be used to produce GPA analogues that could overcome antibiotic resistance. GPA-modifying sulfotransferases are promising tools for generating the unique derivatives. Despite significant sequence and structural similarities, these sulfotransferases modify distinct side chains on the GPA scaffold. To provide insight into the spatial diversity of modifications, we have determined the crystal structure of the ternary complex of bacterial sulfotransferase StaL with the cofactor product 3'-phosphoadenosine 5'-phosphate and desulfo-A47934 aglycone substrate. Desulfo-A47934 binds with the hydroxyl group on the 4-hydroxyphenylglycine in residue 1 directed toward the 3'-phosphoadenosine 5'-phosphate and hydrogen-bonded to the catalytic His67. Homodimeric StaL can accommodate GPA substrate in only one of the two active sites because of potential steric clashes. Importantly, the aglycone substrate demonstrates a flattened conformation, in contrast to the cup-shaped structures observed previously. Analysis of the conformations of this scaffold showed that despite the apparent rigidity due to crosslinking between the side chains, the aglycone scaffold displays substantial flexibility, important for enzymatic modifications by the GPA-tailoring enzymes. We also discuss the potential of using the current structural information in generating unique GPA derivatives.

The influence of the ABO blood type on the distribution of von Willebrand factor in healthy children with no bleeding symptoms.

UNLABELLED: The purpose of this study was to determine the effect of ABO blood groups on von Willebrand factor-ristocetin cofactor activity (vWF-RCo) and on vWF-antigen (vWF-Ag) in children who have no personal or familial history of bleeding. MATERIAL AND METHODS: A survey and testing were performed on 200 children with no personal or familial history of bleeding. In all, 100 of them belonged to blood group O, and the remaining 100 belonged to other blood groups. The blood samples were stored at -80°C for a maximum period of 2 weeks to detect vWF-RCo and vWF-Ag levels. RESULTS: The mean vWF-Ag (± 2 standard deviation [SD]) level in children with blood group O was 86% (± 20%); and for those with non-O blood group, it was 98.8% (± 25%). There was a significant difference between the 2 groups (P < .001). The mean vWF-RCo (± 2 SD) level in children with blood group O was 89% (± 23%); and for those with non-O blood group, it was 103% (± 17%). There was a significant difference between those in the 2 groups (P < .001). The lowest value of vWF-Ag and vWF-RCo levels in children with blood group O was found to be 50%. In conclusion, we showed that the selection of normal ranges based on the ABO group might influence the clinical diagnosis of vWD and that while the approach of using ABO group ranges for a vWF-Ag level lower than 50 IU/dL is scientifically sound, it might not be useful to assist a clinician in identifying people at increased risk of bleeding.

Peptide fragmentation by corona discharge induced electrochemical ionization.

Fundamental studies have greatly improved our understanding of electrospray, including the underlying electrochemical reactions. Generally regarded as disadvantageous, we have recently shown that corona discharge (CD) can be used as an effective method to create a radical cation species [M](+·), thus optimizing the electrochemical reactions that occur on the surface of the stainless steel (SS) electrospray capillary tip. This technique is known as CD initiated electrochemical ionization (CD-ECI). Here, we report on the fundamental studies using CD-ECI to induce analytically useful in-source fragmentation of a range of molecules that complex transition metals. Compounds that have been selectively fragmented using CD-ECI include enolate forming phenylglycine containing peptides, glycopeptides, nucleosides, and phosphopeptides. Collision induced dissociation (CID) or other activation techniques were not necessary for CD-ECI fragmentation. A four step mechanism was proposed: (1) complexation using either Fe in the SS capillary tip material or Cu(II) as an offline complexation reagent; (2) electrochemical oxidation of the complexed metal and thus formation of a radical cation (e.g.; Fe - e(-) → Fe(+·)); (3) radical fragmentation of the complexed compound; (4) electrospray ionization of the fragmented neutrals. Fragmentation patterns resembling b- and y-type ions were observed and allowed the localization of the phosphorylation sites.

Kinetic study of von Willebrand factor self-aggregation induced by ristocetin.

Von Willebrand factor (VWF) is a multimeric glycoprotein present in circulating blood and in secretory granules of endothelial cells and platelets. VWF is sensitive to hydrodynamic shear stress that promotes conformational changes, rendering it able to interact with subendothelial proteins and platelets, thus promoting primary haemostasis. Likewise, the binding of the glycopeptide antibiotic ristocetin to VWF triggers hemostatically relevant conformational transitions. These changes reveal both the interaction site for platelet receptor GpIbalpha and the Tyr1605-Met1606 peptide bond, which is cleaved by the regulatory metalloprotease ADAMTS-13. In this study we investigated by a combined approach of light scattering spectroscopy and turbidimetry the ability of VWF to self-associate in solution in the presence of ristocetin and in the absence of any protein adsorbing surface. Micro- and macro-aggregates induced by ristocetin, have been characterized under static conditions in the early stage of formation and on a longer time scale (up to 10 h). These findings show that VWF multimers form supramolecular structures favoring platelet trapping not only under high shear stress or interaction with external surfaces, but also in solution under static conditions when the conformational state of the protein is changed only by chemical potential of allosteric effectors.