Different pKa Shifts of Internal GLU8 in Human ß-Endorphin Amyloid Revealing a Coupling of Internal Ionization and Stepwise Fibril Disassembly.

As a functional amyloid, human ß-endorphin amyloid fibril features a ß-solenoid conformation and store peptide hormones within acidic secretory granules, which would be released into the blood through fibril disassembly when the cellular milieu pH increases from acidic to neutral level on exocytosis. To gain detailed atomic mechanism of ß-endorphin amyloid fibrils' pH-responsive disassembly, we conduct constant pH molecular dynamics simulations to investigate the structural and dynamical properties of ß-endorphin amyloid fibrils in experiencing the environmental pH changes. Our results demonstrate a clear pKa shift of the internal ionizable residue of GLU8, and this shift becomes even more pronounced when it is buried more deeply in the amyloid fibrils. The unusual pKa of GLU8 reveals that its protonation state changes from the protonated state in the acidic secretory granule to the deprotonated state in the neutral pH conditions in the blood, where the deprotonation of GLU8 leads to unfavorable interactions within the hydrophobic core of the amyloid and subsequent fibril disassembly. The different pKa shifts of GLU8 relative to its positions in the amyloid fibril indicate that the ß-endorphin amyloid fibril disassembly is a stepwise process, accounting for the experimental observation that the disassembly always initiates from the outermost layer. This study reveals the critical role of the protonation state of GLU8 in amyloid fibrils' pH-responsive disassembly, and provides clear insights for understanding the structural transitions of amyloids in hormone secretion. This study also provides theoretical basis for designing pH-sensitive biological tools for specific use with precise positioning of ionizable residues into the hydrophobic interior of proteins.

The three-dimensional structure of human ß-endorphin amyloid fibrils.

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure-function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone ß-endorphin was determined by solid-state NMR. We find that ß-endorphin fibrils are in a ß-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested-and supported with mutagenesis data-that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone ß-endorphin. We find that ß-endorphin fibrils are in a ß-solenoid conformation that is generally reminiscent of other functional amyloids. In the ß-endorphin amyloid, every layer of the ß-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the ß-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of ß-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes.

All-Electronic Quantification of Neuropeptide-Receptor Interaction Using a Bias-Free Functionalized Graphene Microelectrode.

Opioid neuropeptides play a significant role in pain perception, appetite regulation, sleep, memory, and learning. Advances in understanding of opioid peptide physiology are held back by the lack of methodologies for real-time quantification of affinities and kinetics of the opioid neuropeptide-receptor interaction at levels typical of endogenous secretion (<50 pM) in biosolutions with physiological ionic strength. To address this challenge, we developed all-electronic opioid-neuropeptide biosensors based on graphene microelectrodes functionalized with a computationally redesigned water-soluble µ-opioid receptor. We used the functionalized microelectrode in a bias-free charge measurement configuration to measure the binding kinetics and equilibrium binding properties of the engineered receptor with [d-Ala2, N-MePhe4, Gly-ol]-enkephalin and ß-endorphin at picomolar levels in real time.

Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different.

Amyloid polymorphism of twisted and straight ß-endorphin fibrils was studied by negative-stain transmission electron microscopy, scanning transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. Whereas fibrils assembled in the presence of salt formed flat, striated ribbons, in the absence of salt they formed mainly twisted filaments. To get insights into their structural differences at the atomic level, 3D solid-state NMR spectra of both fibril types were acquired, allowing the detection of the differences in chemical shifts of 13C and 15N atoms in both preparations. The spectral fingerprints and therefore the chemical shifts are very similar for both fibril types. This indicates that the monomer structure and the molecular interfaces are almost the same but that these small differences do propagate to produce flat and twisted morphologies at the mesoscopic scale. This finding is in agreement with both experimental and theoretical considerations on the assembly of polymers (including amyloids) under different salt conditions, which attribute the mesoscopic difference of flat versus twisted fibrils to electrostatic intermolecular repulsions.

Heparin acts as a structural component of ß-endorphin amyloid fibrils rather than a simple aggregation promoter.

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide ß-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in the presence of heparin leads to a loss of function. Applying correlative optical super-resolution microscopy methods, we show that heparin incorporates into emerging ß-endorphin fibrils forming an integral component and is essential for amyloid templating. This will have direct implications on ß-endorphin's normal physiological function and raises concerns on the biological relevance of heparin-promoted amyloid models.

The conformational and vibrational behavior of the inhibitory neuropeptide derived from beta-endorphin.

In this study, conformational behavior, structural, and vibrational characterization of the carboxy terminal dipeptide of ß-endorphin (glycy-l-glutamine, glycyl-glutamine, beta-endorphin30-31), which is an inhibitory neuropeptide synthesized from beta-endorphin1-31 in brain stem regions, has been investigated. The theoretically possible stable conformers were searched by means of molecular mechanics method to determine their energetically preferred conformations. The 360 different conformations were calculated with the φ, Ψ, χ dihedral angles using the Ramachandran maps. The most stable conformation of the title molecule is characterized by the extended backbone shape (e) in the BR conformational range with -.78 kcal/mol energy. The cis- and trans-dimeric forms of the dipeptide were also formed and energetically preferred conformations of dimers were investigated. The experimental methods (FT-IR, micro-Raman spectroscopies) coupled with quantum chemical calculations based on density functional theory (DFT) have been used to identify the geometrical, energetic, and vibrational characteristics of the dipeptide. The assignment of the vibrational spectra was performed based on the potential energy distribution of the vibrational modes. To investigate the electronic properties, such as nonlinear optical properties, the electric dipole moment, the mean polarizability, the mean first hyperpolarizability, and HOMO-LUMO energy gaps were computed using the DFT with the B3LYP/6-31++G(d,p) basis set combination. The second-order interaction energies were derived from natural bonding orbital analysis. The focus of this study is to determine possible stable conformation on inhibitory neuropeptide and to investigate molecular geometry, molecular vibrations of monomeric and dimeric forms, and hydrogen bonding interactions of glycy-l-glutamine dipeptide.

17ß-estradiol effect on testicular ß-endorphin expression in Psammomys obesus.

INTRODUCTION: Testicular function in the sand rodent Psammomys obesus is subjected to seasonal alternations with a trigger of spermatogenesis in winter and a total quiescence which extends from late spring to summer. The aim of this study was to investigate the distribution of b-endorphin in the testis at the period of winter sexual activity and at its summer regression, and assess the effect of 17 ß-estradiol treatment on testicular morphology and b-endorphin expression. MATERIAL AND METHODS: The adult males were grouped into 4 groups (rest group, sexually active group, rest treated with 17ß-estradiol group and controls at sexual rest injected with olive oil, n = 5 in each group). Using anti-serum against b-endorphin, we studied its testicular expression by Western blot and cellular location by immunohistochemical (IHC) method, respectively. RESULTS: We detected by Western blot a peptide of 3.5 kDa molecular weight corresponding to b-endorphin only in sexually resting and control males. The 17ß-estradiol treatment induced a clear reduction in the b-endorphin band expression compared with the latter. These results were confirmed by the IHC analysis since b-endorphin was only observed in the testis at sexual rest and in controls, in majority of seminiferous tubules at the level of germ cells. The intensity of IHC labeling was significantly different between spermatogonia and spermatocytes I or round spermatids which revealed the strongest labeling. The intense immunoreactivity was also located in Leydig cells and highly significantly varied compared to the germ cells. The 17 ß-estradiol treatment decreased significantly the ß-endorphin signal in germ cells but not in Leydig cells. CONCLUSION: The 17ß-estradiol treatment induces a repressive effect on seasonal testicular endorphinergic system in P. obesus and this action targets exclusively the germ cells.

Solid-state NMR sequential assignment of the ß-endorphin peptide in its amyloid form.

Insights into the three-dimensional structure of hormone fibrils are crucial for a detailed understanding of how an amyloid structure allows the storage of hormones in secretory vesicles prior to hormone secretion into the blood stream. As an example for various hormone amyloids, we have studied the endogenous opioid neuropeptide ß-endorphin in one of its fibril forms. We have achieved the sequential assignment of the chemical shifts of the backbone and side-chain heavy atoms of the fibril. The secondary chemical shift analysis revealed that the ß-endorphin peptide adopts three ß-strands in its fibril state. This finding fosters the amyloid nature of a hormone at the atomic level.

60 YEARS OF POMC: Lipotropin and beta-endorphin: a perspective.

Many important fields of research had a humble origin. In the distant past, A J P Martin's discovery that amino acids could be separated by paper chromatography and Moore and Stein's use of columns for quantitative amino acid analysis provided the first steps towards the determination of structure in complex biologically active molecules. They opened the door to reveal the essential relationship that exists between structure and function. In molecular endocrinology, for example, striking advances have been made by chemists with their expertise in the identification of structure working with biologists who contributed valuable knowledge and experience. Advantage was gained from the convergence of different background, and it is notable that the whole is greater than the sum. In the determination of structure, it may be recalled that four of the world's great pioneers (Archibald Martin, Rodney Porter, Fred Sanger and Vincent du Vigneaud) were acknowledged for their fundamental contributions when individually they were awarded the Nobel Prize. They foresaw that the identification of structure would prove of outstanding importance in the future. Indeed, study of the structures of ß-endorphin and enkephalin and the different forms of opiate activity they engender has led to a transformation in our understanding of chemical transmission in the brain.

Dynamic Assembly and Disassembly of Functional ß-Endorphin Amyloid Fibrils.

Neuropeptides and peptide hormones are stored in the amyloid state in dense-core vesicles of secretory cells. Secreted peptides experience dramatic environmental changes in the secretory pathway, from the endoplasmic reticulum via secretory vesicles to release into the interstitial space or blood. The molecular mechanisms of amyloid formation during packing of peptides into secretory vesicles and amyloid dissociation upon release remain unknown. In the present work, we applied thioflavin T binding, tyrosine intrinsic fluorescence, fluorescence anisotropy measurements, and solid-state NMR spectroscopy to study the influence of physiologically relevant environmental factors on the assembly and disassembly of ß-endorphin amyloids in vitro. We found that ß-endorphin aggregation and dissociation occur in vitro on relatively short time scales, comparable to times required for protein synthesis and the rise of peptide concentration in the blood, respectively. Both assembly and disassembly of amyloids strongly depend on the presence of salts of polyprotic acids (such as phosphate and sulfate), while salts of monoprotic acids are not effective in promoting aggregation. A steep increase of the peptide aggregation rate constant upon increase of solution pH from 5.0 to 6.0 toward the isoelectric point as well as more rapid dissociation of ß-endorphin amyloid fibrils at lower pH indicate the contribution of ion-specific effects into dynamics of the amyloid. Several low-molecular-weight carbohydrates exhibit the same effect on ß-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, ß-endorphin amyloid fibrils obtained in the presence of heparin demonstrated distinctly different behavior, which we attributed to a dramatic change of the amyloid structure. Overall, the presented results support the hypothesis that packing of peptide hormones/neuropeptides in dense-core vesicles do not necessarily require a specialized cellular machinery.

60 YEARS OF POMC: Purification and biological characterisation of melanotrophins and corticotrophins.

The remarkable conservation of the primary structures and anatomical location of dogfish α-melanocyte-stimulating hormone (MSH), corticotrophin-like intermediate lobe peptide (CLIP) and adrenocorticotrophic hormone (ACTH) compared with mammals reinforced the tissue-specific processing hypothesis of ACTH peptides in the pituitary gland. The cloning of dogfish pro-opiomelanocortin (POMC) led to the identification of δ-MSH and simultaneously revealed the high conservation of the γ-MSH sequence during evolution. These studies have also shown that ß-MSH is much less conserved during evolution and in some species is not even processed from ß-LPH. Human pro-γ-MSH potentiates the corticosteroidogenic activity of ACTH and peptides generated from its N-terminal, in particular big-γ-MSH, appear to have adrenal mitogenic activity. Human big-γ-MSH (from the zona intermedia) may also cause the adrenache. The review finishes with a cautionary note with regard to the misdiagnosis of the ectopic ACTH syndrome in which partial processing of ACTH can result in large concentrations of α-MSH and CLIP, which can interfere in the performance of two-site immunoassays, and the problem of the correct disulphide bridge arrangement in synthetic N-POMC peptides is also discussed.

The second life of antibodies.

Antibodies (immunoglobulins, Ig) are used by the immune system to identify and neutralize foreign objects and are responsible for antigen-binding and effector functions. Immunoglobulin G (IgG) is the major serum immunoglobulin of a healthy human (~75% of the total Ig fraction). The discovery in 1970 of the endogenous tetrapeptide tuftsin (Thr-Lys-Pro-Arg, fragment 289-292 of the C(H2)-domain of the heavy (H) chain of IgG), possessing both immunostimulatory and neurotrophic activities, was an impetus for the search for new biologically active peptides of immunoglobulin origin. As a result, fragments of the H-chain of IgG produced as a result of enzymatic cleavage of IgG within the antigen-antibody complex were discovered, synthesized, and studied. These fragments include rigin (341-344), immunorphin (364-373), immunocortin (11-20), and peptide p24 (335-358) and its fragments. In this review the properties of these peptides and their role in regulating the immune response are analyzed.

Use of 1,5-diaminonaphthalene to combine matrix-assisted laser desorption/ionization in-source decay fragmentation with hydrogen/deuterium exchange.

RATIONALE: In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the deuterons following in-solution hydrogen/deuterium exchange (HDX). This matrix must circumvent the commonly encountered undesired back-exchange reactions, in order to preserve the regioselective deuteration pattern. METHODS: The 1,5-diaminonaphthalene (1,5-DAN) matrix is known to be suitable for MALDI-ISD fragmentation. MALDI Mass Spectrometry Imaging (MSI) was employed to compare 1,5-DAN and other commonly used MALDI matrices with respect to the extent of back-exchange and the uniformity of the H/D exchange profiles within the MALDI spots. We tested the back-exchange on the highly sensitive amyloid-beta peptide (1-40), and proved the regioselectivity on ubiquitin and ß-endorphin. RESULTS: MALDI-MSI results show that 1,5-DAN leads to the least back-exchange over all the spot. MALDI-ISD fragmentation combined with H/D exchange using 1,5-DAN matrix was validated by localizing deuterons in native ubiquitin. Results agree with previous data obtained by Nuclear Magnetic Resonance (NMR) and Electron Transfer Dissociation (ETD). Moreover, 1,5-DAN matrix was used to study the H/D exchange profile of the methanol-induced helical structure of ß-endorphin, and the relative protection can be explained by the polarity of residues involved in hydrogen bond formation. CONCLUSIONS: We found that controlling crystallization is the most important parameter when combining H/D exchange with MALDI. The 1,5-DAN matrix is characterized by a fast crystallization kinetics, and therefore gives robust and reliable H/D exchange profiles using MALDI-ISD.

ß-endorphin via the delta opioid receptor is a major factor in the incubation of cocaine craving.

Cue-induced cocaine craving intensifies, or 'incubates', during the first few weeks of abstinence and persists over extended periods of time. One important factor implicated in cocaine addiction is the endogenous opioid ß-endorphin. In the present study, we examined the possible involvement of ß-endorphin in the incubation of cocaine craving. Rats were trained to self-administer cocaine (0.75 mg/kg, 10 days, 6 h/day), followed by either a 1-day or a 30-day period of forced abstinence. Subsequent testing for cue-induced cocaine-seeking behavior (without cocaine reinforcement) was performed. Rats exposed to the drug-associated cue on day 1 of forced abstinence demonstrated minimal cue-induced cocaine-seeking behavior concurrently with a significant increase in ß-endorphin release in the nucleus accumbens (NAc). Conversely, exposure to the cue on day 30 increased cocaine seeking, while ß-endorphin levels remained unchanged. Intra-NAc infusion of an anti-ß-endorphin antibody (4 µg) on day 1 increased cue-induced cocaine seeking, whereas infusion of a synthetic ß-endorphin peptide (100 ng) on day 30 significantly decreased cue response. Both intra-NAc infusions of the δ opioid receptor antagonist naltrindole (1 µg) on day 1 and naltrindole together with ß-endorphin on day 30 increased cue-induced cocaine-seeking behavior. Intra-NAc infusion of the µ opioid receptor antagonist CTAP (30 ng and 3 µg) had no behavioral effect. Altogether, these results demonstrate a novel role for ß-endorphin and the δ opioid receptor in the development of the incubation of cocaine craving.

Molecular interpretation of ACTH-ß-endorphin coaggregation: relevance to secretory granule biogenesis.

Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and ß-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and ß-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-ß-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-ß-endorphin system, ß-endorphin-only system and ACTH-only system. We find that ß-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with ß-endorphin but also enhances the stability of mixed oligomers of the entire system.

Detection of nonopioid ß-endorphin receptor in the rat myocardium.

Two selective agonists of nonopioid ß-endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high-affinity naloxone-insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [(3)H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled ß-endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [(3)H]immunorphin specific binding with the membranes was inhibited by unlabeled ß-endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α-endorphin, γ-endorphin, [Met(5)]enkephalin and [Leu(5)]enkephalin. Thus, ß-endorphin, immunorphin and octarphin bind to the common high-affinity naloxone-insensitive receptor of the rat myocardial membranes.

Immunostimulating effect of the synthetic peptide octarphin corresponding to ß-endorphin fragment 12-19.

We have synthesized the peptide TPLVTLFK corresponding to ß-endorphin fragment 12-19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [3H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled b-endorphin and the selective agonist of nonopioid b-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled naloxone, a-endorphin, γ-endorphin, or [Met(5)]enkephalin (K(i) > 10 mM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immunocompetent cells in vitro and in vivo: at concentration of 1-10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 µg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.

[Effects and mechanism of action of synthetic peptide octarphin].

We have synthesized the peptide TPLVTLFK corresponding to the ß-endorphin fragment 12-19 (the name given by the authors - octarphin), and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol) and its binding to the murine peritoneal macrophages has been studied. [(3)H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [(3)H]octarphin is inhibited by unlabeled ß-endorphin and selective agonist of non-opioid ß-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM respectively) and not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met(5)]enkephalin (K(i) > 10 µM). Inhibiting activity of unlabeled analogs of octarphin is more then 100 times lower the unlabeled octarphin. Octarphin stimulates activity of murine immunocompetent cells in vitro and in vivo: at the concentration of 1-10 nM enhances the adhesion and spreading of peritoneal macrophages as well as their capacity to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro. Intraperitoneal administration of peptide at dose 20 µg/animal on day 7,3 and 1 prior to the isolation of cells increases activity of peritoneal macrophages as well as T- and B-spleen lymphocytes.

Effect of solvent and electrospray mass spectrometer parameters on the charge state distribution of peptides--a case study using liquid chromatography/mass spectrometry method development for beta-endorphin assay.

Beta-endorphin was used as a model peptide to study the effect of solvent and electrospray mass spectrometer parameters in the optimisation of an assay method for multiply charged compounds using liquid chromatography/mass spectrometry (LC/MS). Unlike with singly charged compounds, the charge state distribution has a significant impact in the method development of multiply charged compounds such as peptides. Using a 50% acetonitrile/water solvent mixture, we found that the ion spray voltage had no influence on the charge state distribution. However, increasing declustering potential led to deprotonation of the higher charge states of the peptide thus causing a shift to lower charge states. The mechanism leading to the deprotonation was examined. It was concluded that the deprotonation is due to endoergic proton transfer from the peptide to solvent molecules clustered to the peptide that occurs in the declustering region. The extent of deprotonation increases with increasing proton affinity of the molecules of the non-aqueous solvent component used. Thus, if desired, deprotonation can be avoided by selecting a low proton affinity solvent such as methanol. The focusing potential was also found to have a great influence on the charge state distribution observed. The results of this study enabled us to select the optimum ion to be used in single ion/reaction monitoring mode. They also provided the most favourable parameter values to be used in the method to obtain the best sensitivity for the ion of choice. The results demonstrate the importance of considering the charge state distribution in the optimisation of electrospray LC/MS methods for multiply charged compounds.

Protein-peptide affinity determination using an h/d exchange dilution strategy: application to antigen-antibody interactions.

A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc.2003, 125, 5252-5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide ß-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.