MS Experiments Research Articles

Efficient identification of raw and ripe tung oil using headspace GC-MS.

To differentiate between the raw type and ripe type of tung oil, it is important to distinguish between the types of tung oil before its application. In the present work, an efficient headspace gas chromatography-mass spectrometry (HS-GC-MS) method was developed for identifying eight samples T1-T8, including the raw tung oil and ripe tung oil. The HS-GC-MS experiments results showed that octanoic acid existed only in ripe tung oil of T2, T4, T6, T8, not in raw tung oil of T1, T3, T5, T7. Combined with structural characterization by tandem mass spectrometry, octanoic acid was screened as an effective marker for distinguishing between raw tung oil and ripe tung oil. Then, the HS-GC-MS method was applied into the putty samples of X1 (raw tung oil with lime) and X2 (ripe tung oil with lime) and successfully identified the samples X1 mixed with raw tung oil and X2 mixed with ripe tung oil. The further validations results suggested that the detection limit of our HS-GC-MS method could reach 1.05 mg/L for octanoic acid, whereas the detection limit of derivative gas chromatography-mass spectrometry (DR-GC-MS) method was 2.74 mg/L for methyl octanoate. The investigation results can also provide the useful information and technical support for the selection of restoration materials and technology in ancient buildings.

Characterization of Forced Degradation Products & Related Substances of Bilastine by Online LC-Q-TOF-MS and In-silico Study of Characterized Compounds

This manuscript is about the identification of degradation products and four related substances of a novel antihistaminic drug Bilastine by LC-Q-TOF-MS/MS. A selective stability-indicating RP-HPLC method was developed for the separation of Bilastine, its 4 related substances and degradation products. Separation was done through Inertsil ODS-3® column (150 x 4.6 mm, 5 µm) using 10 mM ammonium acetate (pH 6.00) and methanol as eluent in a gradient at 1.0 mL/min flow rate. The chromatogram was extracted at 275 nm wavelength. Two degradation products and four related substances were identified and their structures were proposed on the basis of data obtained from LC-Q-TOF-MS/MS experiments. The developed method was validated in accordance with ICH Q2 and USP <1225> guidelines and it was found to be specific, precise, accurate and robust for the intended use. The method was linear in the range of 160-240 µg/mL for drug and 1.15-4.5 µg/mL for related substances with R2 value greater than 0.999 and 0.997 for drug and RS respectively. Percentage of relative standard deviation for validated parameters was less than 2. Structures of the drug, its degradation products and related substances were subjected to in silico studies for prediction of physicochemical, pharmacokinetic and toxicological properties with the help of QikProp module of Schrödinger suite 2017-1 software. Predicted in silico values suggested that they are safe and lie within the recommended range of parameters.

Allocation of Ambipolar Charges on an Organic Diradical with a Vinylene-Phenylenediyne Bridge.

Two redox and magnetically active perchlorotriphenylmethyl (•PTM) radical units have been connected as end-capping groups to a bis(phenylene)diyne chain through vinylene linkers. Negative and positive charged species have been generated, and the influence of the bridge on their stabilization is discussed. Partial reduction of the electron-withdrawing •PTM radicals results in a class-II mixed-valence system with the negative charge located on the terminal PTM units, proving the efficiency of the conjugated chain for the electron transport between the two terminal sites. Counterintuitively, the oxidation process does not occur along the electron-rich bridge but on the vinylene units. The •PTM radicals play a key role in the stabilization of the cationic species, promoting the generation of quinoidal ring segments.

Isolation, Characterization and Antibacterial Activity of ECDY Steroid from Roots of Polycarpaea corymbosa LAM

A new phytoecdysteroid (2- deoxy 20 hydroxy 22 cinnamoyl ecdysone 3, 25 diacetate) was isolated from the roots of Polycarpaea corymbosa Lam (family: caryophyllaceae).The powdered roots were extracted with petroleum ether ( 60-80? C), benzene, ethyl acetate and ethyl alcohol in soxhlet apparatus by simultaneous extraction. The highest yield of benzene extract was chosen for the isolation of ecdysteroid. The white solid pure compound was isolated and tested by TLC and respond positively for Liebermann - burchard reaction. Their structure has been established by UV, IR, 1H, 13C-NMR DEPT -90, DEPT -135 and EI- MS experiments. All the extracts and isolated compound were screened for anti-bacterial activity against B.subtilis, P.vulgaris, E.coli, P.aeruginosa by Kirbybauer method.

Fragmentation reactions of protonated α,ω-diamino carboxylic acids: The importance of functional group interactions.

Protonated members of a homologous series of biologically significant α,ω-diamino carboxylic acids were subjected to collision induced dissociation (CID). The resulting fragmentation patterns were studied using isotopic labeling, quantum mechanical computations, and pseudo MS3 experiments conducted primarily on an ion trap mass spectrometer. Each protonated α,ω-diamino acid showed a primary neutral loss of either ammonia or water; a clear explanation was developed for the observed variation of the two losses within the series. Protonated 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, and 2,7-diaminoheptanoic acid gave secondary losses of water, carbon monoxide, and a loss of water plus carbon monoxide, respectively. In the parallel pathways characterized for the fragmentations of protonated ornithine and lysine, the α-nitrogen of the diamino acid was maintained in the cyclic iminium product formed by successive losses of NH3 and (H2 O + CO), whereas the side-chain nitrogen was retained by consecutive losses of H2 O and (CO, NH3 ). The 1-piperideine ion from protonated lysine was fragmented further, losing ethylene from carbons 4 and 5. Protonated 2,6-diaminopimelic acid fragmented by analogous reactions. Detailed mechanistic schemes for the fragmentation of both protonated 2,3-diaminopropanoic and ornithine were generated from MP2/DFT computations. This work highlights the participation of the side-chain amino group, which distinguishes the gas-phase chemistry of protonated α,ω-diamino acids from the well-documented fragmentation reactions of protonated α-amino acids bearing a hydrogen atom or an alkyl side chain. In general, the results further illustrate the importance of intramolecular separations affecting the specific interactions between functional groups leading to the fragmentation of multifunctional ions.

Selective recognition of bisphenol S isomers in water by β-cyclodextrin

ABSTRACT The two isomers 4,4´-dihydroxydiphenyl sulphone and 2,4´-dihydroxydiphenyl sulphone (bisphenol S, BPS) form inclusion complexes with β-CD in solution, in the gas phase, and in the solid state. The complexes 4,4´-BPS@β-CD and 2,4´-BPS@β-CD were studied in solution by 1D and 2D NMR experiments. Calorimetric investigation by ITC evidenced that the β-CD host shows a greater affinity for 4,4´-BPS, with a 4,4´-BPS/2,4´-BPS selectivity ratio of 6.3. In the gas phase, CID MS experiments indicate that 4,4´-BPS has a higher kinetic barrier to escape from the β-CD cavity than the isomeric 2,4´-BPS. In the solid state, the 4,4´-BPS@β-CD and 2,4´-BPS@β-CD complexes form head-to-head dimers constituted by two β-CD macrocycles, with each β-CD hosting one BPS guest. The dimers of β-CD are sealed by H-bonding interactions involving exclusively the secondary OH groups. Finally, the formation of inclusion complexes between 4,4´-BPS or 2,4´-BPS and β-CD was also followed by FT-IR, DSC, and TGA analysis.

MO1004IMBALANCE IN URINARY PROTEOGLYCANS AND THE INSULIN GROWTH FACTOR (IGF) AXIS IN CHILDREN WITH NEPHROLITHIASIS*

Abstract Background and Aims In vitro experiments have shown that exposure of renal epithelial cells to calcium oxalate crystals leads to synthesis of proteins involved in extracellular matrix (ECM) production. Cell proliferation and accumulation of ECM proteins have been reported in animal models and kidney biopsies from patients with stones. Based on the role of ECM in the Randall plaque formation, we aimed to identify and quantify ECM proteins in the urine of children with nephrolithiasis (RS) using a proteomic approach. Method Prospective, controlled, pilot study of pooled urine from RS (N=30, 24 females, mean age 12.95±4.03 years) versus age- and gender-matched healthy controls (HC), using liquid chromatography-mass spectrometry (LC-MS/MS). Patient inclusion criteria consisted of age 5-18 years, history of clinically (typical renal colic) and radiographically (ultrasound or CT) proven renal stone, at least two 24-hour satisfactory urine collections (urinary creatinine more than 15 mg/kg/day), absence of hematuria or pyuria, and normal renal function. Children who were not toilet trained, or those with bladder stones, nephrocalcinosis, neuropathic bladder, major congenital bladder abnormality, active urinary tract infection, presence of blood in urine, chronic kidney disease, previous major reconstructive bladder surgery requiring catheterization, and significant cardiac, pulmonary, gastro-intestinal, and neurological problems were excluded. Relative protein abundance was estimated using spectral counting. The criteria for protein selection were: 1) patient/control abundance ratios of &amp;gt;5 or &amp;lt;0.2 as a threshold to be well above observed technical variations in MS experiments; and 2) ≤0.05 p-value for the Fisher’s Exact Test. Results were confirmed by ELISA testing. Statistical analyses were conducted with IBM SPSS® version 20. Results We found 36 (15.7%) ECM proteins out of 229 proteins that met the above criteria. Significant differences between RS and HC were found among two proteoglycans and four insulin growth factor (IGF) proteins (Table). Significant increase in the urinary excretion of IGFBP4 in RS (37.00 ± 37.68 ng/mg creatinine ) versus HC (19.04 ± 20.32 ng/mg creatinine) was confirmed by ELISA (p=0.049). Statistically significant correlation was found between urinary IGFBP4 concentration and 24-hour urinary calcium excretion (p&amp;lt;0.001). Conclusion Alteration in proteoglycans and the IGF axis appears to have a significant role in the mechanism of nephrolithiasis, likely by modulating ECM biosynthesis. Further understanding of their roles in nephrolithiasis may aid in generation of novel therapeutic approaches.

Establishment of the tandem mass spectrometric fingerprints of taxane-based anticancer compounds.

Compounds in the taxane drug family are among the most successful and effective chemotherapeutic agents used in the treatment of solid tumors, such as breast, ovarian, and prostate cancers. The tandem mass spectrometric (MS/MS) fragmentation behavior of these compounds is described in detail, and a generalized MS/MS fingerprint is established for the first time. Five compounds, namely paclitaxel, docetaxel, cabazitaxel, cephalomannine, and baccatin III, were evaluated. A hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer was used to obtain accurate mass measurements, whereas MS/MS and second-generation MS/MS (MS3 ) analyses were performed using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were equipped with an electrospray ionization source operated in the positive ion mode. All taxanes showed an abundant singly charged [M + H]+ species in the single-stage analysis with mass accuracies less than 3ppm. The evaluated compounds exhibited common fragmentation behavior in their MS/MS analysis, which allowed for the production of a universal fragmentation pattern. MS3 experiments confirmed the genesis of the various product ions proposed in the fragmentation pathway. In addition, diagnostic product ions were originated from a cleavage in the ester bond between the core diterpene ring structure and the side chain. Varying functional groups present in these compounds resulted in unique product ions that are specific to each structure. The established MS/MS fingerprints will be used in the near future for identification and for the development of multiple reaction monitoring liquid chromatography-MS/MS quantification methods.

Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer.

Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS2) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS2 experiments, following ETD/EThcD MS2 with PTCR MS3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS3.

Transient Response Characteristics and Performance Assessment of a Calorimetric Surface Junction Probe Under Impulsive Thermal Loading

Abstract The measurement of surface temperatures in highly unsteady flow environments is a challenging task pertaining to the need for high-frequency sensors. This paper contains the sequential activities related to a fast response thermal probe (coaxial surface junction probe—CSJP). This probe is prepared in the laboratory, and its salient features are explored for short-duration (∼20 ms) experiments. The surface junction morphology and inside texture of the probe are examined under field emission scanning electron microscope (FESEM). The study confirms the plastic deformation of thermo-elements with an average junction thickness of 21 μm. The static calibration of the probe using a glycerin bath shows a linear trend between voltage and temperature from which the sensitivity value is calculated as 59 μV/°C. The “thermal product” of the probe is also measured experimentally through the “water droplet technique,” and its value is found to be 8677 J/m2 s0.5 K. The concept of one-dimensional heat flux modeling is followed to infer surface heat flux from transient temperatures. For assuring prediction of heat flux, the probe is calibrated experimentally by exposing to a laser source of known wattage (2 W and 3 W). These findings are also supported by numerical simulation of the probe with accuracy in prediction for surface temperature and heat flux as ± 2% and ±3%, respectively. The performance capability of the probe is demonstrated through shock-tube flow experiments to measure instantaneous heat flux. The comparison of the transient response behavior (9500 K/s) with pressure transducer justifies the utility of the probe under hostile flow environment.

Multistimuli-Responsive Fluorescent Organometallic Assemblies Based on Mesoionic Carbene-Decorated Tetraphenylethene Ligands and Their Applications in Cell Imaging

Multistimuli-Responsive Fluorescent Organometallic Assemblies Based on Mesoionic Carbene-Decorated Tetraphenylethene Ligands and Their Applications in Cell Imaging

Separation and identification of impurities from intermediates of istradefylline

伊曲茶碱是一种新型选择性腺苷A2A受体拮抗剂,用于治疗帕金森氏病和改善帕金森氏病初期运动障碍。在伊曲茶碱中间体A1(6-氨基-1,3-二乙基-2,4-(1H,3H)-嘧啶二酮)的合成过程中,碱性条件或高温条件下会伴随生成至少2种副产物,在前期研究中我们已经对该中间体合成过程中的其中一种副产物进行过研究,确定其结构为(E)-N-乙基-2-氰基-3-乙氨基-2-丁烯酰胺。本文采用高效液相色谱(HPLC)对中间体A1的另外一种杂质进行分析:称取0.4 g中间体放入50 mL的烧杯中,依次加入8 mL水、8 mL乙腈,超声溶解,经0.45 μm有机膜过滤,得到测试样品溶液。采用Agilent Zorbax C18色谱柱(150 mm×4.6 mm, 5 μm)分离,柱温35 ℃,流动相为乙腈(A)和水(B),梯度洗脱(tmin/A∶B)=t0/20∶80, t15/60∶40, t20~t50/90∶10;流速1.0 mL/min;检测波长268 nm。然后通过Ceres B制备色谱柱分离,以乙腈-水(30/70, v/v)为流动相,流速为30 mL/min,在268 nm波长下检测,洗脱得到杂质纯品。通过高分辨率质谱(HRMS)、一维核磁共振(NMR)、二维核磁共振(2D NMR)对杂质进行了结构确认,并通过单晶X射线衍射(XRD)进行了表征。杂质经分析确认为1-(1,3-二乙基-2,6-二氧-1,2,3,6-四氢嘧啶-4-基)-3-乙基脲。根据杂质的化学结构推测其生成机理为:在碱性条件或高温条件下合成中间体A1时,过量的二乙基脲继续与中间体A1发生酰胺化反应而得到此副产物。此杂质与伊曲茶碱中间体A1结构相似,会伴随A1参与到伊曲茶碱合成的后续反应中,并对伊曲茶碱的安全性和有效性产生潜在的影响。因此,为了确保伊曲茶碱的质量,在生产过程中需要对该杂质的含量进行控制。

Mammalian Arginase Inhibitory Activity of Methanolic Extracts and Isolated Compounds from Cyperus Species

Polyphenolic enriched extracts from two species of Cyperus, Cyperus glomeratus and Cyperus thunbergii, possess mammalian arginase inhibitory capacities, with the percentage inhibition ranging from 80% to 95% at 100 µg/mL and 40% to 64% at 10 µg/mL. Phytochemical investigation of these species led to the isolation and identification of two new natural stilbene oligomers named thunbergin A-B (1–2), together with three other stilbenes, trans-resveratrol (3), trans-scirpusin A (4), trans-cyperusphenol A (6), and two flavonoids, aureusidin (5) and luteolin (7), which were isolated for the first time from C. thunbergii and C. glomeratus. Structures were established on the basis of the spectroscopic data from MS and NMR experiments. The arginase inhibitory activity of compounds 1–7 was evaluated through an in vitro arginase inhibitory assay using purified liver bovine arginase. As a result, five compounds (1, 4–7) showed significant inhibition of arginase, with IC50 values between 17.6 and 60.6 µM, in the range of those of the natural arginase inhibitor piceatannol (12.6 µM). In addition, methanolic extract from Cyperus thunbergii exhibited an endothelium and NO-dependent vasorelaxant effect on thoracic aortic rings from rats and improved endothelial dysfunction in an adjuvant-induced arthritis rat model.

Binary Classifier for Computing Posterior Error Probabilities in MetaMorpheus.

MetaMorpheus is a free, open-source software program for the identification of peptides and proteoforms from data-dependent acquisition tandem MS experiments. There is inherent uncertainty in these assignments for several reasons, including the limited overlap between experimental and theoretical peaks, the m/z uncertainty, and noise peaks or peaks from coisolated peptides that produce false matches. False discovery rates provide only a set-wise approximation for incorrect spectrum matches. Here we implemented a binary decision tree calculation within MetaMorpheus to compute a posterior error probability, which provides a measure of uncertainty for each peptide-spectrum match. We demonstrate its utility for increasing identifications and resolving ambiguities in bottom-up, top-down, proteogenomic, and nonspecific digestion searches.

New malonylginsenosides from the fresh fruits of Panax notoginseng

Four new malonylginsenosides, malonylnotoginsenoside Fe (1), malonylnotoginsenoside Ra1 (2), malonylgypenoside LXXV (3), and malonylginsenoside Mc (4), together with two known analogues, malonylfloralginsenoside Rc1 (5) and malonylginsenoside Rc (6), were isolated from the fresh fruits of Panax notoginseng. Their structures were determined by MS and NMR experiments. The anti-proliferative activities of the malonylginsenosides (1–6) against SH-SY5Y human neuroblastoma cell line were evaluated using the MTT assay.

What are molecular effects of co-administering vitamin C with artemisinin-type antimalarials? A model mass spectrometry and quantum chemical study

In this study the electrospray ionization mass spectrometry (ESI MS) and quantum chemical modeling methods are employed to examine the interactions of molecules of artemisinin-type drugs and of ascorbic acid (ASC). These biologically significant interactions are relevant to antimalarial therapy, in particular, when artemisinin agents are co-administrated with supporting vitamin/antioxidant preparations or could be affiliated with the patient's food. The formation of stable noncovalent complexes of the artemisinin-type drugs (artemisinin, dihydroartemisinin, α-artemether, and β-arteether) with ascorbic acid molecules in a polar solvent, such as methanol, is revealed by the ESI MS probing of binary systems containing an antimalarial drug and ASC in the 1:1 molar ratio. Also, a peak corresponding to noncovalent [ASC•DPPC•Na]+ cationized complexes is identified in the spectrum of the mixture of ASC and dipalmitoylphosphatidylcholine (DPPC, membrane phospholipid) with the 1:5 molar ratio. Next, the ternary system containing dihydroartemisinin (DHAn; the assumed active metabolite of the artemisinin-type drugs in the human organism), ASC, and DPPC in the 1:1:5 molar ratio is examined. The study reveals a competition between the antimalarial agent and ASC for binding with the DPPC molecules. The existence of the competition is supported by the observation of peaks with similar intensities corresponding to the noncovalent DHAn•DPPC and ASC•DPPC complexes in the mass spectra. An evidence for the complexation between the antimalarial drug and ASC is also found in the spectra of triple model systems studied. To elucidate the structural and energetic characteristics of the noncovalent complexes observed in the ESI MS experiments, model ab initio calculations of DHAn and ASC complexes and clusters of the drug molecules with the polar phosphatidylcholine head of DPPC are performed using the DFT/B3LYP/aug-cc-pVDZ approach. The results of the model study show the possibility of the noncovalent complexation and of the modulation of the biological activity of artemisin-type agents and of the ascorbic acid when they are co-administered in the therapy.

Mapping Binding Epitopes and Allosteric Effects in Staph Enterotoxins

Staphylococcal enterotoxins (SEs) cause food poisoning-like symptoms and can be lethal at microgram amounts. SEs elicit profound immune responses via the bridging of major histocompatibility complex class II (MHCII) molecules on antigen presenting cells with T-cell receptors (TCR). 20B1, 14G8, and 6D3 stand out as promising neutralizing monoclonal antibodies (mAbs) that have been co-crystalized with staphylococcus enterotoxin B (SEB). Although crystal structures have been solved with SEB and a variety of antibodies, it is still not understood why some antibodies effectively neutralize toxins while others do not. Paired with structures of MHC class II and TCR bound to SEB, it has been suggested that 20B1 inactivates SEB by sterically hindering the binding of the toxin to immune molecules. 14G8 and 6D3 also inactivate SEB, but through binding interactions at distal epitopes. Using Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) and X-Ray Footprinting (XRF) in tandem with MS experiments, we observe that neutralizing antibodies binding to distal epitopes elicit allosteric effects in SEB that may drive neutralization.

Isolation, characterization and semi-synthesis of natural products dimeric amide alkaloids

Isolation, characterization and semi-synthesis of natural products dimeric amide alkaloids

Different photocatalytic levels of organics in papermaking wastewater by flocculation-photocatalysis and SBR-photocatalysis: Degradation and GC–MS experiments, adsorption and photocatalysis simulations

Refractory pollutants often exist in wastewater after photocatalysis. Here, experiments, gas chromatography-mass spectrometry (GC–MS) analysis and density functional theory (DFT) simulations were used to explain the different levels of photocatalytic degradation of organics in papermaking wastewater associated with two photocatalytic processes with the goal of improving environmental sustainability. The photocatalytic experiments of flocculation and SBR effluents of papermaking wastewater were studied in a reactor with nano-TiO2 colloid, followed by GC–MS analysis, and the different degradation levels of organics were explained based on DFT adsorption and photocatalysis simulations. The experimental results show that the total COD removal rates were 65.8% and 90.8% for the flocculation-photocatalysis and SBR-photocatalysis processes, and the resin acids and fatty acids in wastewater were classified as easily degradable pollutants and esters as refractory pollutants. The adsorption and photocatalysis simulations show that compared with dehydroabietic acid and pentadecanoic acid, the adsorption of glycerol monostearate by nano-TiO2 colloid is difficult, the reaction pathways for photocatalysis are complex and the band gap of TiO2 photocatalyst after adsorption is wide. The experimental and simulation results indicate that the different degradation levels are attributed to the difficulty of the adsorption and photocatalytic reactions of glycerol monostearate compared with the reactions of the other two easily degradable pollutants. Thus, the different photocatalytic degradation levels of organic pollutants in the two photocatalytic processes of papermaking wastewater indicate that sustainable photocatalytic technology could be applied to different types of industrial wastewater.

Heterologous expression of cryptic biosynthetic gene cluster from Streptomyces prunicolor yields novel bicyclic peptide prunipeptin

Recently, ω-ester-containing peptides (OEPs) were indicated to be a class of ribosomally synthesized and post-translationally modified peptides. Based on genome mining, new biosynthetic gene cluster of OEPs was found in the genome sequence of actinobacterium Streptomyces prunicolor. The biosynthetic gene cluster contained just two genes including precursor peptide (pruA) and ATP-grasp ligase (pruB) coding genes. Heterologous co-expression of the two genes was accomplished using expression vector pET-41a(+) in Escherichia coli. As a result, new OEP named prunipeptin was produced by this system. By site-directed mutagenesis experiment, a variant peptide prunipeptin 15HW was obtained. The bridging pattern of prunipeptin 15HW was determined by combination of chemical cleavage and MS experiments. Prunipeptin 15HW possessed bicyclic structure with an ester bond and an isopeptide bond. The ATP-grasp ligase PruB was indicated to catalyze the two different intramolecular bonds.