Hybrid Linear Trap Quadrupole Orbitrap Research Articles

Using serum peptidomics to discovery the diagnostic marker for different stage of ulcerative colitis

The use of peptidomics to find diagnostic markers has attracted increasing clinical attention. Ulcerative colitis (UC) is a type of inflammatory bowel disease, and the traditional auxiliary diagnostic technique is colonoscopy. However, this invasive method is not effective in distinguishing between patients with endoscopic remission and healthy people, which carries the risk of delayed diagnosis of UC. In this study, we used peptidomics to find serum diagnostic markers for different stages of UC. A total of 78 serum samples were collected to form a training set (60 samples) and a testing set (18 samples). Among them, patients with active UC, remitting UC and healthy people accounted for one third each. The nano-liquid chromatography coupled with hybrid linear trap quadrupole orbitrap mass spectrometry was used for detection of low molecular weight peptides in serum. According to the protein database search and de novo sequencing algorithm, forty peptides were simultaneously identified in all samples. Six biomarker peptides were screened in the training set through orthogonal partial least-squares-discriminant analysis and receiver operating characteristic curve analysis. These six peptides were derived from proteins involved in coagulation and complement activation. We evaluated the diagnostic ability of the six peptides in the testing set through hierarchical cluster analysis, and showed that perturbation of these peptides could distinguish patients with active UC, patients with remitting UC and healthy people. This study validated the feasibility of serum peptidomics for the discovery of diagnostic markers, and provided a potential method for diagnosing different stages of UC.

Characterization and Quantification of Phenolic Constituents in Peach Blossom by UPLC-LTQ-Orbitrap-MS and UPLC-DAD

Peach blossom comes from the flower of Prunus persica (L.) Batsch, which is used as herbal tea and medicine in China and Korea. It could promote defecation and alleviate the abdominal pain. In this paper, the methods, ultra-performance liquid chromatography (UPLC) method coupled with electrospray ionization hybrid linear trap quadrupole orbitrap mass spectrometry (LTQ OrbitrapMS) and UPLC system coupled with a diode array detector (DAD), were developed for the qualitative and quantitative analysis of the flavonoids and phenolic acids in peach blossoms. Eight standards were divided into 3 types according to their basic skeletons: phenolic acids, quercetin-type flavonoids, and kaempferol-type flavonoids. The MSn fragmentation behaviors and diagnostic ions of these 3 types of compounds were proposed to aid the structural identification of components in peach blossom extract. By extracting the diagnostic ions from the mass spectrum in negative mode, a total of 25 compounds, including 8 phenolic acids and 17 flavonoids, were screened out. Among these compounds, 5 compounds (chlorogenic acid, ferulic acid, rutin, hyperoside, and isoquercetrin) were quantitated by UPLC-DAD. The linearity, precision, accuracy, limit of detection, and limit of quantitation were validated for the quantification method. The validated method was applied to assay 9 batches of peach blossoms from different regions. This study was the first report on the systematic qualitative analysis of compounds in peach blossom, providing insights into the quality control of peach blossom.

Identification of In Vivo Metabolites of Dictamnine in Mice Using HPLC-LTQ-Orbitrap Mass Spectrometry.

Dictamnine (4-methoxyfuro[2,3-b]quinolone, DIC), a common furoquinoline alkaloid in the family of Rutaceae, showed diverse biological activities. To investigate the in vivo metabolic pathways of DIC, metabolism of DIC in mice was studied using a high-performance liquid chromatography coupled to electrospray ionization of hybrid linear trap quadrupole orbitrap (HPLC-LTQ-Orbitrap) mass spectrometer. Nine metabolites were identified in the DIC-treated mouse urine, plasma, and fecal samples, of which two were identified as new metabolites. The major metabolic pathways of DIC in animal and human liver microsomes were confirmed in the present study, including o-demethylation, monohydroxylation, N-oxidation, and 2,3-olefinic epoxidation pathways. For the first time, a mono-acetylcysteine conjugate of DIC (M9) was detected from DIC-treated mouse urine and plasma samples, and 4-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (M10) and 2-(2,8-dihydroxy-4-methoxyquinolin-3-yl)acetaldehyde (M11) were identified as new metabolites of DIC; furthermore, using an in vitro human fecal incubation model, furo[2,3-b]quinolin-4-ol (M1) was verified to be a microbial demethylated metabolite of DIC. Collectively, the present study provided new information on the in vivo metabolic fate of DIC.

Characterization of an epoxide-derived metabolite of dictamnine using high-performance liquid chromatography with hybrid linear trap quadrupole orbitrap mass spectrometry.

Dictamnine (4-methoxyfuro[2,3-b]quinolone), a furoquinoline alkaloid of the Rutaceae plant family, has been reported to be a phototoxic and photomutagenic compound, whose exposure can cause carcinogenicity, cytotoxicity, and genotoxicity. Metabolic activation is suggested to play an important role in dictamnine-induced toxicities, and the epoxide metabolite of dictamnine has been reported to be the main intermediate in vitro. The objective of this study was to identify N-acetylcysteine conjugate(s) derived from this reactive dictamnine metabolite in vitro and in vivo. An N-acetylcysteine conjugate of dictamnine was detected in microsomal incubations of dictamnine, as well as bile and urine samples of rats treated with dictamnine. The data obtained from the present work will facilitate the understanding of the mechanism behind dictamnine-induced toxicities.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Identification and quantitative characterization of PSORI-CM01, a Chinese medicine formula for psoriasis therapy, by liquid chromatography coupled with an LTQ Orbitrap mass spectrometer.

PSORI-CM01 is a Chinese medicine formula prepared from medicinal herbs and used in China for the treatment of psoriasis. However, the chemical constituents in PSORI-CM01 have not been clarified yet. In order to quickly define the chemical profiles and control the quality of PSORI-CM01 preparations, ultra-high liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole Orbitrap mass spectrometry (UHPLC-ESI-LTQ/Orbitrap-MS) was applied for simultaneous identification and quantification of multiple constituents. A total of 108 compounds, including organic acids, phenolic acids, flavonoids, and terpenoids, were identified or tentatively deduced on the base of their retention behaviors, MS and MSn data, or by comparing with reference substances and literature data. In addition, an optimized UHPLC-ESI-MS method was established for the quantitative determination of 14 marker compounds in different dosage forms of PSORI-CM01 preparations. The validation of the method, including spike recoveries, linearity, sensitivity (LOQ), precision, and repeatability, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. This is the first report on the comprehensive determination of chemical constituents in PSORI-CM01 preparations by UHPLC-ESI-LTQ/Orbitrap mass spectrometry. The results suggested that the established methods would be a powerful and reliable analytical tool for the characterization of multi-constituents in complex chemical system and quality control of TCM preparations.

Characterization of in vitro metabolites of TM‐2, a potential antitumor drug, in rat, dog and human liver microsomes using liquid chromatography/tandem mass spectrometry

TM-2 (13-(N-Boc-3-i-butylisoserinoyl-4,10-β-diacetoxy-2-α-benzoyloxy-5-β,20-epoxy-1,13-α-dihydroxy-9-oxo-19-norcyclopropa[g]tax-11-ene) is a novel semi-synthetic taxane derivative. Our previous study demonstrated that it is a promising taxane derivative. The in vitro comparative metabolic profile of a drug between animals and humans is a key issue that should be investigated at early stages of drug development to better select drug candidates. In this study, the in vitro metabolic pathways of TM-2 in rat, dog and human liver microsomes were established and compared. TM-2 was incubated with liver microsomes in the presence of NADPH. Two different types of mass spectrometers - a hybrid linear trap quadrupole orbitrap (LC/LTQ-Orbitrap) mass spectrometer and a triple-quadrupole tandem mass spectrometer (LC/QqQ) were employed to acquire structural information of TM-2 metabolites. Accurate mass measurement using LC/LTQ-Orbitrap was used to determine the accurate mass data and elemental compositions of metabolites thereby confirming the proposed structures of the metabolites. For the chemical inhibition study, selective P450 inhibitors were added to incubations to initially characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of TM-2. A total of 12 components (M1-M12) were detected and identified as the metabolites of TM-2 in vitro. M1-M5 were formed by hydroxylation of the taxane ring or the lateral chain. Hydroxylated products can be further oxidized to the dihydroxylated metabolites M6-M10. M11 was a trihydroxylated metabolite. M12 was tentatively identified as a carboxylic acid derivative. The metabolism of TM-2 is much the same in all three species with some differences. The chemical inhibition study initially demonstrated that the formation of M2, the major metabolite of TM-2, is mainly mediated by CYP3A4. Hydroxylation is the major biotransformation of the TM-2 pathway in vitro. CYP3A4 may play a dominant role in the formation of M2 in liver microsomes. The knowledge of the metabolic pathways of TM-2 is important to support further research of TM-2.

Qualitative and quantitative analysis of Rhizoma Smilacis glabrae by ultra high performance liquid chromatography coupled with LTQ OrbitrapXL hybrid mass spectrometry.

Rhizoma Smilacis glabrae, a traditional Chinese medicine (TCM) as well as a functional food, has been commonly used for detoxification treatments, relieving dampness and as a diuretic. In order to quickly define the chemical profiles and control the quality of Smilacis glabrae, ultra high performance liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap mass spectrometry (UHPLC-ESI/LTQ-Orbitrap-MS) was applied for simultaneous identification and quantification of its bioactive constituents. A total of 56 compounds, including six new compounds, were identified or tentatively deduced on the basis of their retention behaviors, mass spectra, or by comparison with reference substances and literature data. The identified compounds belonged to flavonoids, phenolic acids and phenylpropanoid glycosides. In addition, an optimized UHPLC-ESI/LTQ-Orbitrap-MS method was established for quantitative determination of six marker compounds from five batches. The validation of the method, including linearity, sensitivity (LOQ), precision, repeatability and spike recoveries, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. The results suggested that the established method would be a powerful and reliable analytical tool for the characterization of multi-constituent in complex chemical system and quality control of TCM.

Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox (MEQ), 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ's metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N → O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we found that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers

To compare the expression protein profile of seminal plasma from infertile men with oligoasthenoteratozoospermia (OAT) due to oxidative stress with that of healthy, fertile men to determine the proteins indicative of infertility. Experimental study. University hospital and research institute. Semen samples from 11 healthy, fertile (according to the 1999 World Health Organization criteria) male volunteers and 11 infertile idiopathic oligoasthenoteratozoospermia (iOAT) patients. None. Proteomic analysis performed by liquid chromatography mass spectrometry on a hybrid linear trap quadrupole Orbitrap Velos mass spectrometer, carbonylation assay to determine degree of oxidative stress, semiquantitative proteomic analysis, gene ontology, and pathway analysis. A total of 2,489 proteins were identified from seminal plasma, which represents the highest number of unique proteins identified to date. Twenty-four proteins were determined as ≥ 1.5-fold up-regulated in the infertile iOAT males as compared with the fertile controls; and 27 proteins from iOAT patients only were identified as common across all analyses. Only five of the proteins were shared between these two groups. A panel of 46 proteins were identified in patients with iOAT that are potential candidates in understanding the etiology of OAT due to oxidative stress.

Multiple and Sequential Data Acquisition Method: An Improved Method for Fragmentation and Detection of Cross-Linked Peptides on a Hybrid Linear Trap Quadrupole Orbitrap Velos Mass Spectrometer

The identification and validation of cross-linked peptides by mass spectrometry remains a daunting challenge for protein-protein cross-linking approaches when investigating protein interactions. This includes the fragmentation of cross-linked peptides in the mass spectrometer per se and following database searching, the matching of the molecular masses of the fragment ions to the correct cross-linked peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines the speed of the tandem mass spectrometry (MS/MS) duty circle with high mass accuracy, and these features were utilized in the current study to substantially improve the confidence in the identification of cross-linked peptides. An MS/MS method termed multiple and sequential data acquisition method (MSDAM) was developed. Preliminary optimization of the MS/MS settings was performed with a synthetic peptide (TP1) cross-linked with bis[sulfosuccinimidyl] suberate (BS(3)). On the basis of these results, MSDAM was created and assessed on the BS(3)-cross-linked bovine serum albumin (BSA) homodimer. MSDAM applies a series of multiple sequential fragmentation events with a range of different normalized collision energies (NCE) to the same precursor ion. The combination of a series of NCE enabled a considerable improvement in the quality of the fragmentation spectra for cross-linked peptides, and ultimately aided in the identification of the sequences of the cross-linked peptides. Concurrently, MSDAM provides confirmatory evidence from the formation of reporter ions fragments, which reduces the false positive rate of incorrectly assigned cross-linked peptides.

Metabolism of mequindox and its metabolites identification in chickens using LC–LTQ-Orbitrap mass spectrometry

Mequindox (MEQ), 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial and feed additive. Its toxicities have been reported to be closely related to its metabolism. To understand more clearly the metabolic pathways of MEQ, its metabolism in chickens was studied using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC–LTQ-Orbitrap) mass spectrometry. The structures of the MEQ metabolites and their product ions were easily and reliably characterized based on the accurate MS-squared spectra and known structure of MEQ. Twenty-four metabolites were detected in chicken plasma, bile, faeces, and tissues, of which 12 were detected in vivo for the first time. The major metabolic pathways reported previously for in vitro metabolism of MEQ in chicken microsomes were confirmed in this study, including N → O group reduction, carbonyl reduction, and methyl mono-hydroxylation. In addition, deacetylation and acetyl-hydroxylation of MEQ were shown to be important metabolic pathways. Collectively, these data contribute to our understanding of the in vivo metabolism of MEQ.

Qualitative and quantitative characterization of chemical constituents in Xin-Ke-Shu preparations by liquid chromatography coupled with a LTQ Orbitrap mass spectrometer

Xin-Ke-Shu (XKS), a traditional Chinese medicine (TCM) preparation containing five herbal medicines, has been commonly used for the treatment of coronary heart disease in China. However, the chemical constituents in XKS have not been clarified yet. In order to quickly define the chemical profiles and control the quality of XKS preparations, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC–LTQ-Orbitrap) mass spectrometry was applied for simultaneous identification and quantification of multi-constituent. A total of 51 compounds, including phenolic acids, isoflavone- C-glycosides, isoflavone- O-glycosides, flavonoids, and triterpenoid saponins, were identified or tentatively deduced on the base of their retention behaviors, MS and MS n data, or by comparing with reference substances and literatures. In addition, an optimized LC–ESI-MS method was established for quantitative determination of 15 marker compounds in XKS preparations from 7 independent pharmaceutical companies. The validation of the method, including spike recoveries, linearity, sensitivity (LOD and LOQ), precision, and repeatability, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. This is the first report on the comprehensive determination of chemical constituents in XKS preparations by LC–LTQ-Orbitrap mass spectrometry. The results suggested that the established methods would be a powerful and reliable analytical tool for the characterization of multi-constituent in complex chemical system and quality control of TCM preparations.

Online identification of the antioxidant constituents of traditional Chinese medicine formula Chaihu-Shu-Gan-San by LC–LTQ-Orbitrap mass spectrometry and microplate spectrophotometer

Chaihu-Shu-Gan-San (CSGS), a traditional Chinese medicine (TCM) formula containing seven herbal medicines, has been used in treatment of gastritis, peptic ulcer, irritable bowel syndrome and depression clinically. However, the chemical constituents in CSGS had not been studied so far. To quickly identify the chemical constituents of CSGS and to understand the chemical profiles related to antioxidant activity of CSGS, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC–LTQ-Orbitrap) mass spectrometry has been applied for online identification of chemical constituents in complex system, meanwhile, antioxidant profile of CSGS was investigated by the fraction collecting and microplate reading system. As a result, 33 chemical constituents in CSGS were identified. Among them, 13 components could be detected both in positive and in negative ion modes, 20 constituents were determined only in positive ion mode and 2 components were only detected in negative ion mode. Meanwhile, the potential antioxidant profile of CSGS was also characterized by combination of 96-well plate collection of elutes from HPLC analysis and microplate spectrophotometer, in which the scavenging activities of free radical produced by DPPH of each fraction could be directly investigated by the analysis of microplate reader. This study quickly screened the contribution of CSGS fractions to the antioxidant activity and online identified the corresponding active constituents. The results indicated that the combination of LC–MS n and 96-well plate assay system established in this paper would be a useful strategy for correlating the chemical profile of TCMs with their bioactivities without isolation and purification.