蜂毒肽在磷脂膜上取向的高效液相色谱和质谱研究

利用脂质体模型系统研究了跨膜电位对蜂毒肽在磷脂磷上取向的影响,采用高效液相色谱与质谱技术相结合方法,分析了蜂毒肽与磷脂膜作用后的胰蛋白酶酶解产物,结果发现,当蜂毒肽分子与没有跨膜电位的脂质体结合后,它的所有酶切位点都能够酶有效切断,当蜂毒肽与带负向膜电全的脂质体结合后,其Ｎ端的一个酶切位点被封闭,从而说明跨膜电位造成了蜂毒肽在膜上的重瓣取向。     

Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in ∼ 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.     

Quantification of melittin and apamin in bee venom lyophilized powder from Apis mellifera by liquid chromatography-diode array detector-tandem mass spectrometry

A high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method was developed for simultaneous determination of melittin and apamin in crude bee venom lyophilized powder (CBVLP) as the traditional Chinese medicine possessing specific biological activity. Melittin and apamin were extracted with pure water from CBVLP samples followed by HPLC-DAD-MS/MS analysis. The method was validated to demonstrate its selectivity, linearity, limit of quantification (LOQ), intraday precision, interday precision, accuracy, recovery, matrix effect, and stability. The assay was linear over the concentration ranges of 1-100 and 0.2-25 μg/ml with limit of quantifications (LOQs) of 1.0 and 0.3 μg/ml for melittin and apamin, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 2.2% to 11.4% for intraday repeatability and from 3.2% to 13.1% for interday intermediary precision. The concentrations of endogenous melittin and apamin in CBVLP samples ranged from 46% to 53% and from 2.2% to 3.7% of dry weight, respectively. This rapid, simple, precise, and sensitive method allowed the simultaneous determination of melittin and apamin to evaluate authenticity and quality of CBVLP samples.     

Acyl Transfer from Membrane Lipids to Peptides Is a Generic Process

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography–mass spectrometry analysis of peptide–lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH 7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3 h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.     

Rapid determination of finasteride in human plasma by UPLC–MS/MS and its application to clinical pharmacokinetic study

A rapid, specific, and sensitive method utilizing reversed-phase ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated to determine finasteride levels in human plasma. The plasma samples were prepared by liquid–liquid extraction with ethyl acetate, evaporation, and reconstitution. MS/MS analyses were performed on a triple–quadrupole tandem mass spectrometer by monitoring protonated parent → daughter ion pairs at m/z 373 → 305 for finasteride and m/z 237 → 194 for carbamazepine (internal standard, IS). The method was validated with respect to linearity, recovery, specificity, accuracy, precision, and stability. The method exhibited a linear response from 0.1 to 30 ng/mL (r² > 0.998). The limit of quantitation for finasteride in plasma was 0.1 ng/mL. The relative standard deviation (RSD) of intra- and inter-day measurements was less than 15% and the method was accurate within −6.0% to 2.31% at all quality-control levels. The mean extraction recovery was higher than 83% for finasteride and 84% for the IS. Plasma samples containing finasteride were stable under the three sets of conditions tested and the processed samples were stable up to 29 h in an autosampler at 5 °C. Detection and quantitation of both analytes within 3 min make this method suitable for high-throughput analyses. The method was successfully applied to a pharmacokinetic study of finasteride in healthy volunteers following oral administration.     

Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study

Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. d-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of d-amphetamine in drug research and its interest in toxicologic–forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of d-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of d-amphetamine in rat blood using MS³ scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC–MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1 mm × 30 mm, 3.5 μm) using gradient elution at a flow rate of 1.0 mL/min over a 2 min run time. An Applied Biosystems API4000 QTRAP™ mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS³ scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1 → 119.1 → 91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1 → 208.2. The linear dynamic range was established over the concentration range 0.5–1000 ng/mL (r² = 0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS³) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (K_a) of 2.85 × 10⁶ M^− 1 and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (ΔG⁰), − 8.8 kcal mol^− 1, obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

Development of an LC-MS/MS method for the simultaneous quantification of aripiprazole and dehydroaripiprazole in human plasma

A selective, sensitive, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of aripiprazole and its active metabolite dehydroaripiprazole in human plasma has been developed using papaverine as internal standard (IS). LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI⁺) and selected reaction monitoring (SRM). The assays for aripiprazole and dehydroaripiprazole were linear over the ranges of 0.1 to 600 ng/ml and 0.01 to 60 ng/ml, respectively. The average recoveries in plasma samples both were better than 85%. The intra- and interrun precision and accuracy values were found to be within the assay variability criteria limits according to the US Food and Drug Administration guidelines. The developed method was proved to be suitable for use in a clinical pharmacokinetic study after a single oral administration of a 5-mg aripiprazole tablet in healthy Chinese volunteers.     

Determination of free and glucuronidated kaempferol in rat plasma by LC–MS/MS: Application to pharmacokinetic study

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid–liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm × 2.1 mm, 4.5 μm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min⁻¹. The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.     

Development of a high-throughput method for the determination of ethosuximide in human plasma by liquid chromatography mass spectrometry

A simple, rapid, sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the quantification of ethosuximide in human plasma is described. Analyte was chromatographed on a Hypersil Gold C₁₈ column (100 mm × 2.1 mm, i.d., 1.9 μm) with isocratic elution at a flow rate of 0.250 mL/min and pravastatin was used as the internal standard. The assay involves a simple solid-phase extraction procedure of 0.25 mL human plasma and the analysis was performed on a triple-quadrupole tandem mass spectrometer by MRM mode via electrospray ionization (ESI). The method was linear in the concentration range of 0.25–60.0 μg/mL. The lower limit of quantification (LLOQ) was 0.25 μg/mL. The within- and between-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 95.1% and 94.4% for ethosuximide and pravastatin, respectively. The analysis time for each sample was 1.8 min. The method was highly reproducible and gave peaks with excellent chromatography properties.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Identification of N-terminal acetylation of recombinant human prothymosin α in Escherichia coli

Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin α is an essential protein in immune stimulation and apoptosis regulation. The protein is N-terminal acetylated in eukaryotes, but similar modification has never been found in recombinant protein produced in prokaryotes. In this study, two mass components of recombinant human prothymosin α expressed in Escherichia coli were identified and separated by RP-HPLC. Mass spectrometry of the two components showed that one of them had a 42 Da mass increment as compared with the theoretical mass of human prothymosin α, which suggested a modification of acetylation. The mass of another one was equal to that of the theoretical one. Peptides mass spectrometry of the modified component showed that the 42-Da mass increment occurred in the N-terminal peptide domain, and MS/MS peptide sequencing of the N-terminal peptide found that the acetylated modification occurred at the N-terminal serine residue. So, part of the recombinant human prothymosin α produced by E. coli was N-terminal acetylated. This finding adds a new clue for the mechanism of acetylated modification in prokaryotes, and also suggested a new method for production of N-terminal modificated prothymosin α and thymosin α1.     

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3 M trehalose or liposome solution, then cooled with slow (0.95 ± 0.02 °C/min), medium (73 ± 3 °C/min) and fast (265 ± 12 °C/min) cooling rates and storage in liquid nitrogen, followed by a 37 °C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66% ± 5% vs 29% ± 4%) compared to control RBCs (c-RBC, p = 0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p = 0.039 and p = 0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p = 0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p = 0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.     

A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M]⁺ ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K]⁺ and [M + Na]⁺ were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.     

Improved liquid chromatography–tandem mass spectrometry method for the analysis of eptifibatide in human plasma

A rapid, selective and highly sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C₁₈ column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6 → m/z 646.4 and m/z 931.6 → m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1–1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within ±7.6% of nominal values. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 μg/kg bolus of eptifibatide to 8 healthy volunteers.     

Structural analysis of a highly acetylated protein using a curved-field reflectron mass spectrometer

Matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry (MS/MS) were used to determine the multiple acetylation sites in the histone acetyltransferase (HAT): p300-HAT. Partial cleavage of the peptides containing acetylated lysine residues by trypsin provided a set of nested sequences that enabled us to determine that multiple acetylation occurs on the same molecule. At the same time, cleavages resulting in a terminal unacetylated lysine suggested that not all of these sites are fully modified. Using MS and MS/MS, we were able to characterize both the unmodified and acetylated tryptic peptides covering more than 82% of the protein.     

A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry

Chemokines are secreted proteins that play roles in inducing chemotaxis, extravasation, and activation of leukocytes associated with inflammatory or homeostatic processes. Tyrosine sulfation of the chemokine receptor has been shown to be important for binding and signaling. We have applied a mass spectrometry method to measure the contribution of this posttranslational modification to binding of its ligand chemokine. Using nano-electrospray time-of-flight mass spectrometry (nano-ESI TOF MS), we determined the association constants of C-C motif chemokine 7 (CCL7) with C-C chemokine receptor type 2 (CCR2), monosulfated CCR2, and disulfated CCR2 peptides to be 1.1 × 10⁴ M⁻¹, 3.9 × 10⁴ M⁻¹, and 4.0 × 10⁵ M⁻¹, respectively. To our knowledge, this is the first reported association constant measurement between a protein and sulfated peptide using MS. Furthermore, nano-ESI MS was used to characterize noncovalent binding interactions among CCL7, Arixtra (a pentasaccharide glycosaminoglycan [GAG] analog), and disulfated CCR2 peptide. A lack of observable ternary complex formation prompted investigation of competitive binding. Results of this study suggest that CCR2 competes partially with GAG for CCL7 binding and that disulfated CCR2 peptide has a higher binding affinity than Arixtra, which correlates with data from association constant measurements for CCL7-disulfated CCR2 and CCL7-Arixtra.