Scan MS Research Articles

A comprehensive UPLC-MS(/MS) based approach for screening and confirmation of illegally added erectile dysfunction (ED) drugs in natural health products

The adulteration of herbal/dietary supplements with erectile dysfunction (ED) drugs and their analogues is reported worldwide and is an increasing problem. The sale of so-called 100%, ’all-natural’ products has become a highly profitable business for online pharmacies, however these products can pose a serious threat to consumers owing to the undisclosed presence of approved/prescription drugs or the unknown safety and toxicity profile of unapproved ED drugs. The Drug QC Laboratory, Qatar, has been involved in the testing of adulterated and counterfeit products for a number of years. A simple and rapid UPLC-MS/MS procedure for the analysis of 42 synthetic ED compounds in herbal supplements is presented. A spectral library for the compounds and their analogues was generated from reference standards for automated routine analysis. Full scan MS analysis was performed simultaneously in both positive and in high energy negative ESI modes; In addition a MS/MS method was developed for confirmation and quantification of compounds. The method was applied to 87 suspected dietary products which were received to our laboratory. Twenty three were found to be adulterated: 11 with sildenafil, four with thiodimethylsildenafil/thiosildenafil, two with dapoxetine and five contained a combination of verdenafil, tadalafil, aminotadalafil, hydroxthiohomo sildenafil. One sample contained unknown analogue named as propoxyphenyl thiohomosildenafil. Developed method is a novel screening method that is suitable for both the detection of known and unknown ED drugs and their analogues. This is the first description of a single analytical method with this capability.

Identification of Unanticipated and Novel N-Acyl L-Homoserine Lactones (AHLs) Using a Sensitive Non-Targeted LC-MS/MS Method.

N-acyl L-homoserine lactones (AHLs) constitute a predominant class of quorum-sensing signaling molecules used by Gram-negative bacteria. Here, we report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and quantitate known, unanticipated, and novel AHLs in microbial samples. Using a hybrid quadrupole-high resolution mass analyzer, this method integrates MS scans and all-ion fragmentation MS/MS scans to allow simultaneous detection of AHL parent-ion masses and generation of full mass spectra at high resolution and high mass accuracy in a single chromatographic run. We applied this method to screen for AHL production in a variety of Gram-negative bacteria (i.e. B. cepacia, E. tarda, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, and R. sphaeroides) and discovered that nearly all of them produce a larger set of AHLs than previously reported. Furthermore, we identified production of an uncommon AHL (i.e. 3-oxo-C7-HL) in E. carotovora and P. stewartii, whose production has only been previously observed within the genera Serratia and Yersinia. Finally, we used our method to quantitate AHL degradation in B. cepacia, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, the non-AHL producer E. coli, and the Gram-positive bacterium B. subtilis. We found that AHL degradation ability varies widely across these microbes, of which B. subtilis and E. carotovora are the best degraders, and observed that there is a general trend for AHLs containing long acyl chains (≥10 carbons) to be degraded at faster rates than AHLs with short acyl chains (≤6 carbons).

Untargeted screening of unknown xenobiotics and potential toxins in plasma of poisoned patients using high-resolution mass spectrometry: Generation of xenobiotic fingerprint using background subtraction

A novel analytical workflow was developed and applied for the detection and identification of unknown xenobiotics in biological samples. High-resolution mass spectrometry (HRMS)-based data-independent MSE acquisition was employed to record full scan MS and fragment spectral datasets of test and control samples. Then, an untargeted data-mining technique, background subtraction, was utilized to find xenobiotics present only in test samples. Structural elucidation of the detected xenobiotics was accomplished by database search, spectral interpretation, and/or comparison with reference standards. Application of the workflow to analysis of unknown xenobiotics in plasma samples collected from four poisoned patients led to generation of xenobiotic profiles, which were regarded as xenobiotic fingerprints of the individual samples. Among 19 xenobiotics detected, 11 xenobiotics existed in a majority of the patients' plasma samples, thus were considered as potential toxins. The follow-up database search led to the tentative identification of azithromycin (X5), α-chaconine (X9) and penfluridol (X12). The identity of X12 was further confirmed with its reference standard. In addition, one xenobiotic component (Y5) was tentatively identified as a penfluridol metabolite. The remaining unidentified xenobiotics listed in the xenobiotic fingerprints can be further characterized or identified in retrospective analyses after their spectral data and/or reference compounds are available. This HRMS-based workflow may have broad applications in the detection and identification of unknown xenobiotics in individual biological samples, such as forensic and toxicological analysis and sport enhancement drug screening.

Wide-scope screening of pesticides in fruits and vegetables using information-dependent acquisition employing UHPLC-QTOF-MS and automated MS/MS library searching

This paper presents an application of ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for simultaneous screening and identification of 427 pesticides in fresh fruit and vegetable samples. Both full MS scan mode for quantification, and an artificial-intelligence-based product ion scan mode information-dependent acquisition (IDA) providing automatic MS to MS/MS switching of product ion spectra for identification, were conducted by one injection. A home-in collision-induced-dissociation all product ions accurate mass spectra library containing more than 1700 spectra was developed prior to actual application. Both qualitative and quantitative validations of the method were carried out. The result showed that 97.4% of the pesticides had the screening detection limit (SDL) less than 50μg kg-1 and more than 86.7% could be confirmed by accurate MS/MS spectra embodied in the home-made library. Meanwhile, calibration curves covering two orders of magnitude were performed, and they were linear over the concentration range studied for the selected matrices (from 5 to 500μg kg-1 for most of the pesticides). Recoveries between 80 and 110% in four matrices (apple, orange, tomato, and spinach) at two spiked levels, 10 and 100μg kg-1, was 88.7 or 86.8%. Furthermore, the overall relative standard deviation (RSD, n = 12) for 94.3% of the pesticides in 10μg kg-1 and 98.1% of the pesticides in 100μg kg-1 spiked levels was less than 20%. In order to validate the suitability for routine analysis, the method was applied to 448 fruit and vegetable samples purchased in different local markets. The results show 83.3% of the analyzed samples have positive findings (higher than the limits of identification and quantification), and 412 commodity-pesticide combinations are identified in our scope. The approach proved to be a cost-effective, time-saving and powerful strategy for routine large-scope screening of pesticides.

Fragmentation Patterns of Aromatic 2,5-diketopiperazines Using Liquid chromatography/Mass Spectrometry

Diketopiperazines (DKPs) are cyclic dipeptides which have been detected in a variety of natural products, especially in thermally treated or fermented foods and beverages, providing a metallic bitter taste. DKPs, mainly due to their characteristic heterocyclic system, have been reported to exhibit a broad spectrum of biological activities including antimicrobial, antiviral, antitumor, antihyperglycaemic and antimutagenic. DKPs are formed from unprotected linear dipeptides under basic conditions especially if the necessary head-to-tail folding is not prevented by steric constraints. A methodology to identify DKPs in complex matrices is of high importance. Up to now, only a few studies have reported the MS fragmentation patterns on a certain number of DKPs. The purpose of this study was to develop a liquid chromatography method for separation and identification of DKPs in complex matrices (i.e. food and beverages) as well as a high resolution mass spectrometry method providing accurate full scan MS and MSn data in order to investigate the fragmentation pattern and confirm the chemical formula and structure of DKPs containing aromatic amino acids. Our results which were supported by in silico DFT energy calculations, revealed different and common fragmentation pathways as well as a series of characteristic fragment ions which are representative of the amino acid residues. The role of MSn was signified by the results since it provided a clear picture of the fragmentation cascades. The obtained fragments are diagnostic and could be used to distinguish cyclic dipeptides in different matrices. Keywords: 2, 5-diketopiperazines, cyclic dipeptides, LC-MSn, fragmentation pattern, bioactive compounds.

Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling

In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin.

Metabolic profile modifications in milk after enrofloxacin administration studied by liquid chromatography coupled with high resolution mass spectrometry

High resolution accurate mass spectrometry (HRMS) operating in full scan MS mode was used in the search and identification of metabolites in raw milk from cows medicated with enrofloxacin. Data consisting of m/z features were taken throughout the entire chromatogram of milk samples from medicated animals and were compared with blank samples. Twenty six different compounds were identified. Some of them were attributed to structures related to enrofloxacin while others were dipeptides or tripeptides. Additionally, enrofloxacin was administered in a controlled treatment for three days. Milk was collected daily from the first day of treatment and until four days after in the search for the identified compounds. The obtained data were chemometrically treated by Principal Component Analysis. Samples were classified by this method into three different groups corresponding to days 1–2, day 3 and days 4–7 considering the different concentration profile evolution of metabolites during the days studied. Tentative metabolic pathways were designed to rationalize the presence of the newly identified compounds.

Multiplexed data independent acquisition (MSX-DIA) applied by high resolution mass spectrometry improves quantification quality for the analysis of histone peptides.

We present the MS-based application of the innovative, although scarcely exploited, multiplexed data-independent acquisition (MSX-DIA) for the analysis of histone PTMs. Histones are golden standard for complexity in MS based proteomics, due to their large number of combinatorial modifications, leading to isobaric peptides after proteolytic digestion. DIA has, thus, gained popularity for the purpose as it allows for MS/MS-based quantification without upfront assay development. In this work, we evaluated the performance of traditional DIA versus MSX-DIA in terms of MS/MS spectra quality, instrument scan rate and quantification precision using histones from HeLa cells. We used an MS/MS isolation window of 10 and 6 m/z for DIA and MSX-DIA, respectively. Four MS/MS scans were multiplexed for MSX-DIA. Despite MSX-DIA was programmed to perform two-fold more MS/MS events than traditional DIA, it acquired on average ∼5% more full MS scans, indicating even faster scan rate. Results highlighted an overall decrease of background ion signals using MSX-DIA, and we illustrated specific examples where peptides of different precursor masses were co-fragmented by DIA but not MSX-DIA. Taken together, MSX-DIA proved thus to be a more favorable method for histone analysis in data independent mode.

Stepped collisional energy MS(All) : an analytical approach for optimal MS/MS acquisition of complex mixture with diverse physicochemical properties.

The analysis of complex mixtures is becoming increasingly important in various fields, such as nutrition, medicinal plants and metabolomics. The components contained in such complex mixtures are always characterized with diverse physiochemical properties that pose a major challenge during the optimization of various parameters using liquid chromatography-mass spectrometer (LC-MS). The parameter 'CE energy' that is normally set at a fixed value with a moderate range of CE spread during data-dependent acquisition (DDA) analysis, a prevalent approach for untargeted identification, often fails to generate sufficient MS/MS fragment ions for untargeted identification of components from complex mixtures. Here we developed a simple and generally applicable acquisition method named stepped MS(All) (sMS(All) ) in this study, aiming to obtain optimal MS/MS spectra for identification of chemically diverse compounds from complex mixtures. sMS(All) collects serial MS(All) scans acquired at low CE to gradually ramped-up high CE values in a cycle that conventional DDA scans cannot afford. The resultant MS/MS spectra of each compound were compared and evaluated among serial MS(All) scans, and the optimal spectra were used for identification. An untargeted data analysis strategy was then employed to analyze these optimal MS/MS spectra by searching common diagnostic ions and connecting the diagnostic ion families into a network via bridging components. This sMS(All) -based route enables identification of 71 natural products from a herbal preparation, whereas only 53 out of 71 compounds were identified using the classical DDA approach. Therefore, the sMS(All) -based approach is expected to find its wide applications for characterization of vastly diverse compounds with no priori knowledge from various complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous Detection of 46 Veterinary Drug Residues in Animal Meat by UHPLC

A novel UHPLC–MS/MS approach, using multi-period multiple-reaction monitoring (mpMRM), was developed for the detection of 46 veterinary drugs including representative beta-agonists, quinolones, sulfonamides, tetracyclines in food of animal origin samples. A simple procedure based on protein precipitation associated with ultrafiltration was employed to realize a generic sample preparation for food of animal origin. In particular, multi-period multiple-reaction monitoring (mpMRM) was developed as a MS scan mode for veterinary drugs to meet the requirements of qualitative and quantitative detection of multiple veterinary drug residues and improve sensitivity and reliability in this method. Furthermore, the method was fully validated for all 46 drugs, and the specificity, linearity, limit of detection, limit of quantification, accuracy and precision results are good. Using the developed method, 30 pork samples and 20 beef samples were analysed, and the result is consistent with that using GB methods (National standard method of the People’s Republic of China) but using less time, which demonstrates that our developed method is not only accurate and reliable, but also greatly improves the cost-effectiveness of the analytical procedures. This approach not only is much more effective than the traditional sample preparation method, but also provides data on four classes of veterinary drugs in one analytical run, which enabled high-throughput and efficiency. The obtained results proved the usefulness of the method as applied in the field of residue analysis.

Comprehensive analysis of 61 characteristic constituents from Siraitiae fructus using ultrahigh-pressure liquid chromatography with time-of-flight mass spectrometry

A systematic method was established for the qualitative and quantitative analysis of characteristic components such as triterpenoids and flavonols in Siraitiae fructus, a famous antidiabetic traditional Chinese medicine (TCM). For qualitative analysis, 61 characteristic components were identified using the ultra-high performance liquid chromatography coupled with photo-diode array and quadrupole/time-of-flight mass spectrometry (UPLC-PDA-QTOF-MS/MS) based on a multiple product ions filtering (mPIF) strategy, of which 22 compounds were characterized for the first time from Siraitiae fructus. For quantitative detection, a relative quantitation assay using an extract ion chromatogram (EIC) of the full scan MS experiment was validated and employed to assess the quantity of the 61 identified compounds in 40 batches Siraitiae fructus samples from different sources. Additionally, the principal component analysis (PCA) indicated that 40 samples could be clustered into four groups and the cultivated variety was an important factor for sample classification. The methods used in present study might be also valuable for simultaneous investigation of multiple components from Siraitiae fructus for the purpose of holistic quality control, phytochemistry and metabolic studies.

Comprehensive untargeted lipidomic analysis using core–shell C30 particle column and high field orbitrap mass spectrometer

The goal of untargeted lipidomics is to have high throughput, yet comprehensive and unambiguous identification and quantification of lipids. Novel stationary phases in LC separation and new mass spectrometric instruments capable of high mass resolving power and faster scanning rate are essential to achieving this goal. In this work, 4 reversed phase LC columns coupled with a high field quadrupole orbitrap mass spectrometer (Q Exactive HF) were thoroughly compared using complex lipid standard mixture and rat plasma and liver samples. A good separation of all lipids was achieved in 24min of gradient. The columns compared include C30 and C18 functionalization on either core–shell or totally porous silica particles, with size ranging from 1.7 to 2.6μm. Accucore C30 column showed the narrowest peaks and highest theoretical plate number, and excellent peak capacity and retention time reproducibility (<1% standard deviation). As a result, it resulted in 430 lipid species identified from rat plasma and rat liver samples with highest confidence. The high resolution offered by the up-front RPLC allowed discrimination of cis/trans isomeric lipid species, and the high field orbitrap mass spectrometer afforded the clear distinction of isobaric lipid species in full scan MS and the unambiguous assignment of sn-positional isomers for lysophospholipids in MS/MS. Taken together, the high efficiency LC separation and high mass resolving MS analysis are very promising tools for untargeted lipidomics analysis.

Application of a high-resolution benchtop quadrupole-Orbitrap mass spectrometry for the pharmacokinetics and tissue distribution of Danhong injection in guinea pig

Danhong injection (DHI) is a compound preparation of traditional Chinese medicine Salviae miltiorrhiza (a.k.a. Danshen) and Flos Carthami tinctorii. Phenolic acids, such as salvianic acid A sodium (SAS), protocatechualdehyde (PAL), salvianolic acid B (SAB) and flavonoids, such as rutin, are the main bioactive ingredients of DHI. This paper presents an application of ultra high-performance liquid chromatography and quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HR MS) to quantify salvianic acid A sodium, protocatechualdehyde, salvianolic acid B, and rutin in guinea pig plasma and tissues for pharmacokinetic and tissue distribution studies. Biological samples were processed with methanol extraction, and calibration curves were determined with indometacin as internal standard (I.S.). The analytes were separated by a Hypersil GOLD aQ C18 column and detected using negative ion full MS/dd-MS2 (data-dependent MS2) mode. The full MS scan acquired data for identification and quantification, and dd-MS2 scan obtained product ion spectra for confirmation. The response showed good linear relationship over the concentration range between 10.22 and 5110ng/mL for salvianic acid A sodium, 10.06–5030ng/mL for salvianolic acid B, 11.12–5560ng/mL for protocatechualdehyde, and 10.58–5290ng/mL for rutin, all the coefficients of correlation (r2) >0.9990. The lower limit of quantification (LLOQ) was 10ng/mL for the analytes. Overall, the novel UHPLC-Q-Orbitrap has demonstrated great performance for rapidity, accuracy, high sensitivity, and high selectivity. It was successfully applied to the pharmacokinetic and tissue distribution studies of Danhong injection. These acquired data would provide helpful information for the clinical applications, mechanism studies, and technology promotion of DHI in the future.

Metabolic profiling for the identification of Huntington biomarkers by on-line solid-phase extraction capillary electrophoresis mass spectrometry combined with advanced data analysis tools.

In this work, an untargeted metabolomic approach based on sensitive analysis by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild-type (wt) and HD (R6/1) mice of different ages (8, 12, and 30 weeks), were analyzed by C18 -SPE-CE-MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS datasets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow-up the HD progression. The intracellular signaling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors, and changed expression of neurotransmitters.

Identification and Quantification of the Major Constituents in Egyptian Carob Extract by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

Background:Carob - Ceratonia siliqua L., commonly known as St John's-bread or locust bean, family Fabaceae - is one of the most useful native Mediterranean trees. There is no data about the chromatography methods performed by high performance liquid chromatography (HPLC) for determining polyphenols in Egyptian carob pods.Objective:To establish a sensitive and specific liquid chromatography–electrospray ionization (ESI)-tandem mass spectrometry (MSn) methodology for the identification of the major constituents in Egyptian carob extract.Materials and Methods:HPLC with diode array detector and ESI-mass spectrometry (MS) was developed for the identification and quantification of phenolic acids, flavonoid glycosides, and aglycones in the methanolic extract of Egyptian C. siliqua. The MS and MSn data together with HPLC retention time of phenolic components allowed structural characterization of these compounds. Peak integration of ions in the MS scans had been used in the quantification technique.Results:A total of 36 compounds were tentatively identified. Twenty-six compounds were identified in the negative mode corresponding to 85.4% of plant dry weight, while ten compounds were identified in the positive mode representing 16.1% of plant dry weight, with the prevalence of flavonoids (75.4% of plant dry weight) predominantly represented by two methylapigenin-O-pentoside isomers (20.9 and 13.7% of plant dry weight).Conclusion:The identification of various compounds present in carob pods opens a new door to an increased understanding of the different health benefits brought about by the consumption of carob and its products.SUMMARY This research proposed a good example for the rapid identification of major constituents in complex systems such as herbs using sensitive, accurate and specific method coupling HPLC with DAD and MS, which facilitate the clarification of phytochemical composition of herbal medicine for better understanding of their nature and biological activities. Abbreviation used: HPLC: High performance liquid chromatography, DAD: Diode array detector, MS: Mass spectrometry, TR: Retention time.

Synchronized Desorption Electrospray Ionization Mass Spectrometry Imaging.

Desorption electrospray ionization (DESI) has emerged as a powerful technique for mass spectral analysis and imaging under ambient conditions. Synchronization of DESI (sDESI) with the ion injection period (IT)of low-duty cycle mass spectrometers has been previously shown to improve sensitivity and reduce the amount of sample depleted during the acquisition of each spectrum (viz. MS scan time). In this report, we describe the development and characterization of an sDESI mass spectrometry imaging source (sDESI-MSI). Our results show that synchronization of DESI with the IT of an LTQ Orbitrap-XL mass spectrometer improves spatial resolution by factors of ∼4-6. In addition, under certain experimental conditions, synchronization was essential to acquire distinct MS images of low-intensity endogenous FAs (< 5% relative intensity) in fingermarks at high sampling frequencies (step sizes ≤ 75 μm). The magnitudes of these improvements in performance depend on the properties of the microdroplet spray, sample, and surface. Simulations that model analyte movement during desorption and the "washing effect" replicate the experimental results with the washing parameter having the greatest impact on performance. Thus, synchronization improves spatial resolution and sensitivity by decreasing the percentage of the total MS scan time that analytes are influenced by the "washing effect". Generally, synchronization of DESI with IT improves performance and expands the range of analytes, surfaces, and experimental conditions amenable to DESI-MSI, especially for analytes that are weakly attached to a surface.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

Mass-spectrometric fragmentation of sodium 2-(4-methyl-5-(thiophene-2-yl)-4H-1,2,4-triazole-3-ylthio)acetate

The study of physical and chemical characteristics and establishing patterns of mass spectrometric fragmentation are the actual tasks of modern pharmaceutical science, have both scientific interest and practical importance.Aim. The purpose of our experiment was to confirm the identity and study patterns of mass spectrometric decomposition for sodium 2-(4-methyl-5-(thiophene-2-yl) -4H-1,2,4-triazoles-3-ilthio) acetate.Methods and results. The study was carried out on the apparatus LC MS / MS: LTQ Orbitrap. After completing MS scan, it has been established that the maximum peak in the chromatogram corresponds protonated ion 2-(4-methyl-5-(thiophene-2-yl)-4H-1,2,4-triazoles-3-ylthio)acetic acid m/z 256.0208, and empirical formula test compound - C9H9N3O2S2. MS/MS MH+ analysis showed that protonated molecule of the 2-(4-methyl-5-(thiophene-2-yl)-4H-1,2,4-3-triazole-3-ylthio)acetic acid under voltage in contact with molecules of helium in the collision cell was disintegrated from m/z 256.0208 into some fragments. The first time the mass spectrometric investigation sodium 2-(4-methyl-5-(thiophene-2-yl)-4H-1,2,4-triazoles-3-ylthio) acetate has been undertaken.Conclusions. It has been established that the fragmentation of the molecule initially occurs with the dissociation of water molecule from carboxyl group. Under the influence of gas the next to come off may be a carboxyl group. In course of further dissociation the bond between the sulfur atom and acetic residue is destroyed or disruption of a covalent bond between sulfur and carbon of 1,2,4-triazole occurs.

Low Resolution Data-Independent Acquisition in an LTQ-Orbitrap Allows for Simplified and Fully Untargeted Analysis of Histone Modifications.

Label-free peptide quantification in liquid chromatography-mass spectrometry (LC-MS) proteomics analyses is complicated by the presence of isobaric coeluting peptides, as they generate the same extracted ion chromatogram corresponding to the sum of their intensities. Histone proteins are especially prone to this, as they are heavily modified by post-translational modifications (PTMs). Their proteolytic digestion leads to a large number of peptides sharing the same mass, while carrying PTMs on different amino acid residues. We present an application of MS data-independent acquisition (DIA) to confidently determine and quantify modified histone peptides. By introducing the use of low-resolution MS/MS DIA, we demonstrate that the signals of 111 histone peptides could easily be extracted from LC-MS runs due to the relatively low sample complexity. By exploiting an LTQ-Orbitrap mass spectrometer, we parallelized MS and MS/MS scan events using the Orbitrap and the linear ion trap, respectively, decreasing the total scan time. This, in combination with large windows for MS/MS fragmentation (50 m/z) and multiple full scan events within a DIA duty cycle, led to a MS scan cycle speed of ∼45 full MS per minute, improving the definition of extracted LC-MS chromatogram profiles. By using such acquisition method, we achieved highly comparable results to our optimized acquisition method for histone peptide analysis (R(2) correlation > 0.98), which combines data-dependent acquisition (DDA) and targeted MS/MS scans, the latter targeting isobaric peptides. By using DIA, we could also remine our data set and quantify 16 additional isobaric peptides commonly not targeted during DDA experiments. Finally, we demonstrated that by performing the full MS scan in the linear ion trap, we achieve highly comparable results as when adopting high-resolution MS scans (R(2) correlation 0.97). Taken together, results confirmed that histone peptide analysis can be performed using DIA and low-resolution MS with high accuracy and precision of peptide quantification. Moreover, DIA intrinsically enables data remining to later identify and quantify isobaric peptides unknown at the time of the LC-MS experiment. These methods will open up epigenetics analyses to the proteomics community who do not have routine access to the newer generation high-resolution MS/MS generating instruments.

On-Line Synthesis and Analysis by Mass Spectrometry

In this laboratory experiment, students learn how to use ESI to accelerate chemical synthesis and to couple it with on-line mass spectrometry for structural analysis. The Hantzsch synthesis of symmetric 1,4-dihydropyridines is a classic example of a one-pot reaction in which multiple intermediates can serve to indicate the progress of the reaction as a function of the electrospray variable parameters. This reaction allows students to gauge the effects of the spray variables while observing the enhanced reaction rate through product analysis in the charged microdroplets. Soft ionization techniques used in conjunction with collision-induced dissociation provide students experience with full scan MS to infer reaction progress and collision-induced fragmentation patterns for structural analysis. The experiment as a whole combines the physical phenomena of electrospray, Hantzsch chemical reactivity, and analytical measurements involving use of an ion trap mass spectrometer.