Intact glucosinolate analysis in plant extracts by programmed cone voltage electrospray LC/MS: performance and comparison with LC/MS/MS methods

We present a comprehensive, sensitive, and highly specific negative ion electrospray LC/MS method for identifying all structural classes of glucosinolates in crude plant extracts. The technique is based on the observation of simultaneous maxima in the abundances of the m/z 96 and 97 ions, generated by programmed cone voltage fragmentation, in the mass chromatogram. The abundance ratios lie in the range 1:2-1:4 ([m/z 96]/[m/z 97]). Examination of the corresponding full-scan mass spectra allows individual glucosinolates of all structural classes to be identified rapidly and with confidence. The use of linearly programmed cone voltage fragmentation enhances characteristic fragment ions without compromising the abundance of the analytically important [M - H]- ion and its associated (and analytically useful) sulfur isotope peaks. Detection limits are in the low nanogram range for full-scan, programmed cone voltage spectra. Comparison of the technique with LC/MS/MS methods (product ion, precursor ion, and constant neutral loss scans) has shown that the sensitivity and selectivity of the programmed cone voltage method is superior. Data obtained on a variety of plant extracts confirmed that the methodology was robust and reliable.     

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0–3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Arbutin derivatives from the buds of Vaccinium dunalianum and their MS/MS spectrometric fragmentations

Four arbutin derivatives were isolated from the buds of Vaccinium dunalianum in which 4-hydroxyphenyl-6′-(3''-O-β-D-glucopyranosyl-4''-hydroxycinnamoyl)-O-β-D-glucopyranoside (1) was a new compound. The structure of the new compound was determined on the basis of NMR and HR-ESI-MS data. All the arbutin derivatives were subjected to the MS/MS analyses from which the MS/MS spectrometric fragmentations were summarized.     

FragmentationAnalyzer: An open‐source tool to analyze MS/MS fragmentation data

A thorough understanding of the fragmentation processes in MS/MS can be a powerful tool in assessing the resulting peptide and protein identifications. We here present the freely available, open‐source FragmentationAnalyzer tool ( http://fragmentation‐analyzer.googlecode.com ) that makes it straightforward to analyze large MS/MS data sets for specific types of identified peptides, using a common set of peptide properties. This enables the detection of fragmentation pattern nuances related to specific instruments or due to the presence of post‐translational modifications.     

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC₅₀=78.38±0.15) which suggests its antioxidant potential.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

Relationship Between Phenolic Content Determined by LC/MS/MS and Antioxidant Capacity and Enzyme Inhibition of Cyclotrichium niveum L.

Cyclotrichium niveum (Boiss.) Manden & Scheng belonging to the Lamiaceae family, which is an endemic species in the eastern Anatolian region of Turkey, has an important place in terms of ethno-botany. The phytochemical composition of the plant, inhibition of acetylcholinesterase (AChE) (which hydrolyzes the neurotransmitter acetylcholine), inhibition of paraoxonase for antiatherosclerotic activity (hPON 1) (which detoxifies organophosphates), and antioxidant capacity were all investigated in this study. Phytochemical content was determined by LC/MS/MS, and enzyme inhibition and antioxidant capacity studies were determined by spectrophotometer. Antioxidant capacity of C. niveum extracts (methanol, hexane, and water) was determined by applying ABTS⋅⁺, DPPH⋅, FRAP, and CUPRAC methods. Both the water and the methanol extracts of the C. niveum exhibited significant inhibition on the AChE (IC₅₀ value for methanol and water extract 0.114±0.14 mg/mL (R2:0.997) and 0.178±0.12 mg/mL (R²: 0.994), respectively). In contrast, the methanol and water extracts of the C. niveum did not exhibit the inhibition effect on hPON 1. The highest activity for ABTS⋅⁺ was 66.53 % in the water extract, and DPPH⋅ was 55.03 % in the methanol extract. In the metal-reducing power assay, the absorbance was 0.168±0.04 for FRAP water extract and 0.621±0.01 for CUPRAC methanol extract. According to LC/MS/MS analyses, hydroxybenzoic acid, salicylic acid, syringic acid, acetohydroxamic acid and luteolin determined in the plant extract. As a consequence, C. niveum which has antioxidant, anti-atherogenic and anti-neurodegenerative properties has the potential to be used as a natural medication instead of synthetic drugs used in Alzheimer's patients.     

Multiplexed LC‐MS/MS analysis of horse plasma proteins to study doping in sport

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC‐MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race‐horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis‐driven discovery of doping biomarker candidates.     

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants—terrestrial manatee relatives—were targeted. These included 5α-reduced progestins (5α-pregnane-3,20-dione [5α-DHP] and 3α-hydroxy-5α-pregnan-20-one [5α-P3-OH]) and 17α-hydroxyprogesterone (17α-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20α-hydroxyprogesterone (20α-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5α-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5α-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17α-OHP metabolite was not detected in any tested female. The 20α-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.     

Long term anoxia in rainbow trout investigated by 2-DE and MS/MS

Twenty-four hours of N(2) induced anoxia induced global perturbations on protein expression in rainbow trout hypodermal fibroblasts cell line. Anoxia was obtained by depleting the medium of O(2) by flushing with N(2), and protein changes were studied by 2-DE coupled with MS providing quantitative measurements of a large number of proteins in one single study. The anoxic insult changed the level of 33 protein spots: 22 of these were up-regulated compared to the control situation and 11 were down-regulated. Using MS/MS sequencing 19 of the 33 protein spots that changed were identified, corresponding to a success rate of more than 50%. The identified proteins included two proteins involved in energy metabolism namely phosphoglycerate mutase and isocitrate dehydrogenase. In addition we observed the up-regulation of a cluster of proteins that contribute to cytoskeleton function. These are calpain, EB1, and Rho GDP dissociation inhibitor (GDI). The up-regulation of Rho GDI was shown to develop in a time dependent manner with no significant increase for up to 8 h of anoxia. In conclusion, this study provides a thorough investigation of the effect of anoxia in a cell line from rainbow trout.     

水生植物轮藻特殊气味的分析研究

本实验采用热脱附气质联用技术(TCT-GC/MS)对山西省内三种轮藻的挥发性气味的化学成分(VOCs)进行了分析,共鉴定出43种物质,主要为烷烃类、酮类、醇类等物质,其中野生不连续轮藻、实验室培养的不连续轮藻和普生轮藻的挥发性物质组成基本相似,以烷烃类为主,而钝节拟丽藻的气味组成与它们存在较大差异,说明其存在属间差异;在轮藻的VOCs组成中,发现了苯并噻唑、青叶醛、α-蒎烯和为薄荷烯等有记载的对昆虫起趋避和杀灭作用的化合物,所以轮藻应具有杀虫活性;另外还在其VOCs组成中发现了苯系物的存在,说明轮藻对其有吸附作用,但这种吸附作用同样存在属间差异。     

Selective analysis of phenolic compounds in propolis by HPLC-MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy. Copyright (c) 2007 John Wiley & Sons, Ltd.     

纳升电喷雾串联质谱技术对阻断泛素通路诱导凋亡相关蛋白质的鉴定

应用毛细管液相色谱 电喷雾 四极杆 飞行时间串联质谱和纳升电喷雾 四极杆 飞行时间串联质谱技术 ,对阻断白血病细胞泛素通路诱发的凋亡相关蛋白质进行了鉴定。通过双向电泳发现 ,蛋白质斑点H在阻断Mo7e白血病细胞泛素通路 2h之后 ,表达量明显增加 ,6h达到最高。该斑点经MALDI TOF MS肽质量指纹谱分析未获结果 ,但通过上述 2种串联质谱技术获得其胰蛋白酶水解肽段的串联质谱图和肽段的全长序列 ,经检索均确认为RhoGDIβ蛋白。进一步发现阻断泛素通路还诱发了另 2个斑点出现 ,位于H点附近 ,经鉴定为同一蛋白质 ,可能是不同翻译后修饰所造成     

Quantitative Detection of 15 Serum Bile Acid Metabolic Products by LC/MS/MS in the Diagnosis of Primary Biliary Cholangitis

To determine 15 bile acid metabolic products in human serum by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and value their diagnostic outcome in primary biliary cholangitis (PBC). Serum from 20 healthy controls and 26 patients with PBC were collected and went LC/MS/MS analysis of 15 bile acid metabolic products. The test results were analyzed by bile acid metabolomics, and the potential biomarkers were screened and their diagnostic performance was judged by statistical methods such as principal component and partial least squares discriminant analysis and area under curve (AUC). 8 differential metabolites can be screened out: Deoxycholic acid (DCA), Glycine deoxycholic acid (GDCA), Lithocholic acid (LCA), Glycine ursodeoxycholic acid (GUDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurodeoxycholic acid (TDCA), Glycine chenodeoxycholic acid (GCDCA). The performance of the biomarkers was evaluated by the AUC, specificity and sensitivity. In conclusion, DCA, GDCA, LCA, GUDCA, TLCA, TUDCA, TDCA and GCDCA were identified as eight potential biomarkers to distinguish between healthy people and PBC patients by multivariate statistical analysis, which provided reliable experimental basis for clinical practice.     

GOAT – A simple LC‐MS/MS gradient optimization tool

Modern nano‐HPLC systems are capable of extremely precise control of solvent gradients, allowing high‐resolution separation of peptides. Most proteomics laboratories use a simple linear analytical gradient for nano‐LC‐MS/MS experiments, though recent evidence indicates that optimized non‐linear gradients result in increased peptide and protein identifications from cell lysates. In concurrent work, we examined non‐linear gradients for the analysis of samples fractionated at the peptide level, where the distribution of peptide retention times often varies by fraction. We hypothesized that greater coverage of these samples could be achieved using per‐fraction optimized gradients. We demonstrate that the optimized gradients improve the distribution of peptides throughout the analysis. Using previous generation MS instrumentation, a considerable gain in peptide and protein identifications can be realized. With current MS platforms that have faster electronics and achieve shorter duty cycle, the improvement in identifications is smaller. Our gradient optimization method has been implemented in a simple graphical tool (GOAT) that is MS‐vendor independent, does not require peptide ID input, and is freely available for non‐commercial use at http://proteomics.swmed.edu/goat/     

Development and validation of a spectral library searching method for peptide identification from MS/MS

A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30,000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching.