High-resolution Mass Analysis Research Articles

Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionation Using Solid-Phase Extraction Cartridges.

Discovery-driven comparative proteomics employing the bottom-up strategy with label-free quantification on high-resolution mass analyzers like an Orbitrap in a hybrid instrument has the capacity to reveal unique biological processes in the context of plant metabolic engineering. However, proteins are very heterogeneous in nature with a wide range of expression levels, and overall coverage may be suboptimal regarding both the number of protein identifications and sequence coverage of the identified proteins using conventional data-dependent acquisitions without sample fractionation before online nanoflow liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). In this chapter, we detail a simple and robust method employing high-pH reversed-phase (HRP) peptide fractionation using solid-phase extraction cartridges for label-free proteomic analyses. Albeit HRP fractionation separates peptides according to their hydrophobicity like the subsequent nanoflow gradient reversed-phased LC relying on low pH mobile phase, the two methods are orthogonal. Presented here as a protocol with yeast (Saccharomyces cerevisiae) as a frequently used model organism and hydrogen peroxide to exert cellular stress and survey its impact compared to unstressed control as an example, the described workflow can be adapted to a wide range of proteome samples for applications to plant metabolic engineering research.

Design, synthesis and biological evaluation of 1,2,4-oxadiazole linked 1,2,4-thiadiazole-pyrimidines as anticancer agents

Abstract A novel library of 1,2,4-oxadiazole linked 1,2,4-thiadiazole-pyrimidines (10a-j) were designed and synthesized. All compounds were characterized by FTIR, NMR and high resolution Mass analysis. Further, these compounds were screened for their anticancer activity against a panel of human cancer cell lines such as MCF-7 (human breast cancer), A-549 (human non -small cell lung cancer), Colo-205 (human adenocarcinoma colon cancer) and A2780 (human ovarian cancer) by employing MTT assay. The clinical drug (etoposide) used as positive control. Among all synthesized compounds, five compounds 10b, 10c, 10d, 10i and 10j were possessed more promising anticancer activity on all cell lines.

Models for prehistoric lithic quarry development

Archaeologists have struggled to find meaningful and cost effective ways to analyze and interpret prehistoric quarry sites. Quarry site deposits typically contain large quantities of broken rock fragments that generally lack morphological attributes commonly used for lithic debitage analysis. Remnants of quarry pits often overlap and converge, forming complex cultural landscapes that are difficult to document and understand. Archaeological excavations near Glens Falls, New York, uncovered evidence of prehistoric mine pits, dense layers of chert quarry debris, and intensively battered bedrock surfaces. High resolution mass analysis was used to analyze large quantities of quarry debris. A new classification for prehistoric quarry sites and new models for quarry site development are proposed.

Semiautomated glycoproteomics data analysis workflow for maximized glycopeptide identification and reliable quantification.

Glycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly evolving in many research areas, leading to a demand in reliable data analysis tools. In recent years, several bioinformatic tools were developed to facilitate and improve both the identification and quantification of glycopeptides. Here, a selection of these tools was combined and evaluated with the aim of establishing a robust glycopeptide detection and quantification workflow targeting enriched glycoproteins. For this purpose, a tryptic digest from affinity-purified immunoglobulins G and A was analyzed on a nano-reversed-phase liquid chromatography–tandem mass spectrometry platform with a high-resolution mass analyzer and higher-energy collisional dissociation fragmentation. Initial glycopeptide identification based on MS/MS data was aided by the Byonic software. Additional MS1-based glycopeptide identification relying on accurate mass and retention time differences using GlycopeptideGraphMS considerably expanded the set of confidently annotated glycopeptides. For glycopeptide quantification, the performance of LaCyTools was compared to Skyline, and GlycopeptideGraphMS. All quantification packages resulted in comparable glycosylation profiles but featured differences in terms of robustness and data quality control. Partial cysteine oxidation was identified as an unexpectedly abundant peptide modification and impaired the automated processing of several IgA glycopeptides. Finally, this study presents a semiautomated workflow for reliable glycoproteomic data analysis by the combination of software packages for MS/MS- and MS1-based glycopeptide identification as well as the integration of analyte quality control and quantification.

Thiol-ene microfluidic chip for fast on-chip sample clean-up, separation and ESI mass spectrometry of peptides and proteins

Mass spectrometry (MS) is a key technology for sensitive and high-resolution mass analysis of peptides and proteins. Sample clean-up and chromatographic separation is typically performed prior to MS analysis to limit adduct formation and ionization suppression. Usually, this requires a high-pressure LC pump system equipped with expensive metal chromatographic columns placed in-line of an electrospray ionization (ESI) source. Microfluidic devices coupled to MS have gained considerable attention, due to the promise of low manufacturing costs, low sample consumption and channels with a high surface area to volume ratio and tailorable functional groups. Here, we describe a thiol-ene microfluidic chip capable of fast chromatographic sample clean-up, concentration, and separation of complex protein and peptide mixtures with direct on-chip ESI. On-chip reversed-phase chromatography (RPC) was performed through an in-situ polymerized monolith frit for retaining inexpensive commercially available reversed-phase (RP) spherical particles, while on-chip ESI is achieved through an emitter monolithically implemented by precision micro milling. The on-chip integration of both RPC and ESI emitter allowed for a minimization of dead-volumes and enables very fast sample clean-up, efficient ionization, and mass analysis of peptides and proteins from complex matrices.

Naphthoquinone-Based Meroterpenoids from Marine-Derived Streptomyces sp. B9173.

Naphthoquinone-based meroterpenoids are hybrid polyketide-terpenoid natural products with chemical diversity and a broad range of biological activities. Here, we report the isolation of a group of naphthoquinone-containing compounds from Streptomyces sp. B9173, and their structures were elucidated by using a combination of spectroscopic techniques, including 1D, 2D NMR, and high-resolution mass (HRMS) analysis. Seven flaviogeranin congeners or intermediates, three of which were new, have been derived from common naphthoquinone backbone and subsequent oxidation, methylation, prenylation, and amino group incorporation. Both flaviogeranin B1 (1) and B (2) contain an amino group which was incorporated into the C8 of 1,3,6,8-terhydroxynaphthalene (THN). Flaviogeranin D (3) contains an intact C-geranylgeranyl residue attached to the C2 of THN, while the O-geranylgeranyl group of 2 links with the hydroxyl on the C2 site of THN. Four compounds were selected and tested for antibacterial activity and cytotoxicity, with 3 and flaviogeranin C2 (5) displaying potent activity against selected bacteria and cancer cell lines. In light of the structure features of isolated compounds and the biosynthetic genes, a biosynthetic pathway of naphthoquinone-based flaviogeranins has been proposed. These isolated compounds not only extend the structural diversity but also represent new insights into the biosynthesis of naphthoquinone-based meroterpenoids.

Characterization of nonvolatile polar compounds from Brazilian oils by electrospray ionization with FT-ICR MS and Orbitrap-MS

Among the different types of available high-resolution mass spectrometers, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and Orbitrap-MS are important tools for the study of nonvolatile crude oil fraction composition. No in deep comparison between each analyzer was still performed, mostly with a wide broad of real crude oil samples. Therefore, in this study a representative set of 30 Brazilian crude oils with a wide range of American Petroleum Institute (API) gravity (13.1–53.7) were considered. The ICR and Orbitrap analyzers allowed the elemental composition to be obtained by accurate mass measurements, and the most abundant species were mainly N, N2, O3, O1, O2, NO2, NS, NOS, and OS classes. In general, FT-ICR MS favored the detection of higher molecular weight analytes in relation to Orbitrap-MS. The notable distribution of the heteroatom classes showed that crude oils with intermediate API gravity presented a higher relative abundance of nitrogenous compounds (Nx), while lighter oils presented a higher abundance of oxygenated compounds (O1). The heavier ones had a higher abundance of O2 class, which was not verified by Orbitrap-MS, where a prevalence of O2 class was detected in major crude oils. An increase in average molar weight values was observed when we changed technique (Orbitrap-MS → FT-ICR MS) and ionization mode (negative → positive), since FT-ICR MS detected substances with higher m/z. In this way, this work examines the application of two different high-resolution mass analyzers, and shows the versatility and analytical response of each one within petroleomics.

Studies of the Fragmentation Mechanisms of Deprotonated Lignin Model Compounds in Tandem Mass Spectrometry.

Unlabeled and deuterium-labeled dimeric lignin model compounds with β-O-4 linkages were evaporated and ionized using negative ion mode electrospray ionization, transferred into a linear quadrupole ion trap, isolated, and subjected to collision-activated dissociation (CAD; MS2 experiments). The elemental compositions of the fragment ions were determined by using a high-resolution Orbitrap mass analyzer, and their structures were examined using further CAD experiments (MSn experiments wherein n = 2-5). Data analysis was facilitated by determining the fragmentation pathways for several deprotonated model compounds. The structures of the key fragment ions of several pathways were determined by comparison of the CAD mass spectra measured for undeuterated and deuterated analogues and for deprotonated authentic compounds. Some of the proposed reaction mechanisms were tested by examining additional deprotonated synthetic model compounds. Quantum chemical calculations were used to delineate the most likely reaction pathways and reaction mechanisms. This work provides basic information needed for the design of tandem mass spectrometry-based CAD sequencing strategies for mixtures of lignin degradation products.

Classification and Authentication of Paprika by UHPLC-HRMS Fingerprinting and Multivariate Calibration Methods (PCA and PLS-DA).

In this study, the feasibility of non-targeted UHPLC-HRMS fingerprints as chemical descriptors to address the classification and authentication of paprika samples was evaluated. Non-targeted UHPLC-HRMS fingerprints were obtained after a simple sample extraction method and C18 reversed-phase separation. Fingerprinting data based on signal intensities as a function of m/z values and retention times were registered in negative ion mode using a q-Orbitrap high-resolution mass analyzer, and the obtained non-targeted UHPLC-HRMS fingerprints subjected to unsupervised principal component analysis (PCA) and supervised partial least squares regression-discriminant analysis (PLS-DA) to study sample discrimination and classification. A total of 105 paprika samples produced in three different regions, La Vera PDO and Murcia PDO, in Spain, and the Czech Republic, and all of them composed of samples of at least two different taste varieties, were analyzed. Non-targeted UHPLC-HRMS fingerprints demonstrated to be excellent sample chemical descriptors to achieve the authentication of paprika production regions with 100% sample classification rates by PLS-DA. Besides, the obtained fingerprints were also able to perfectly discriminate among the different paprika taste varieties in all the studied cases, even in the case of the different La Vera PDO paprika tastes (sweet, bittersweet, and spicy) which are produced in a very small region.

An arsenal of tools based on Kendrick mass defects to process congested electrospray ionization high-resolution mass spectra of polymers with multiple charging.

Electrospray ionization (ESI) favors the multiple charging of high molecular weight polymer samples and allows their high-resolution mass analysis in the low-mass range. It also induces the detection of numerous ion series at different charge states with different adducts complicating the interpretation of the mass spectrum which should be facilitated by an appropriate data processing. An arsenal of tools based on the Kendrick mass defect (KMD) is proposed to process congested ESI high-resolution mass spectra of poly(propylene oxide) (PPO) samples. The combination of regular, charge-dependent, and resolution-enhanced KMD plots in addition to a "remainders" plot and a new three-dimensional plot offers unrivaled capabilities of filtering for any minor series among thousands of points. The sequential data processing is conducted using Kendo, a spreadsheet developed in-house for an advanced KMD analysis. The charge-state distribution is easily evaluated by counting the parallel lines in a regular KMD plot. A charge-dependent resolution-enhanced KMD plot instantly reveals the variation of adducted ions at a given charge state, helping the user to choose the best analytical conditions. Ion series at different charge states from PPO oligomers carrying different end-groups are also efficiently extracted using several combinations of KMD and remainders plots and assigned using a new simulator tool. The innovative combination of existing and new KMD-related plots, selection tools, and simulator all combined in a single spreadsheet dramatically facilitates the processing and interpretation of complex ESI mass spectral data. The presented tools may be extended to any other class of homo-, co- and terpolymers.

Cobalt complexes containing salen-type pyridoxal ligand and DMSO for cycloaddition of carbon dioxide to propylene oxide

Cobalt complexes containing a salen-type pyridoxal ligand with pyridine were synthesized as a new Co(III) catalytic system for the cycloaddition of carbon dioxide. Two cobalt(III) complexes possessing a salen-type pyridoxyl ligand were synthesized by the reaction of pyridoxal ligands (pyr2en = (N,N′-bis(pyridoxylideneiminato)ethylene) and pyr2cy = (N,N′-bis(pyridoxylideneiminato)cyclohexane)) and Co(OAc)2 and characterized by various analytical methods, including infrared spectroscopy and high-resolution mass analysis. Single-crystal X-ray crystallography analysis confirmed that the cobalt pyr2en complex had a distorted octahedral structure: the tetradentate Schiff base ligand binds the cobalt metal in one plane, and the metal center adopts an octahedral geometry by the additional coordination of acetate and dimethyl sulfoxide. The synthesized complexes were used as catalysts in the cycloaddition of carbon dioxide (CO2) to propylene oxide. The catalysts showed high activity for cycloaddition between CO2 and epoxides, even at a low loading (0.5 mol%), in the presence of various cocatalysts.

Enhanced Sequence Coverage of Large Proteins by Combining Ultraviolet Photodissociation with Proton Transfer Reactions.

Ultraviolet photodissociation (UVPD) produces rich and informative fragmentation of intact protein ions, but in the case of high mass proteins (>30 kDa) the spectra are congested with overlapping isotope patterns of highly charged fragment ions. In the most congested regions, many fragments cannot be confidently identified even when high-resolution mass analyzers and modern deconvolution algorithms are used. Gas-phase ion-ion proton transfer reactions (PTR), which reduce the charge states of highly charged ions, can be used to alleviate this congestion and facilitate the identification of additional fragment ions when performed following UVPD. We have developed protocols for sequentially performing PTR on multiple populations of ions generated by UVPD in a way that can be tailored to balance the depth of characterization with speed and throughput. The improvements in sequence coverage and fragment identifications are demonstrated for four proteins ranging in size from 29 to 56 kDa. Sequence coverages up to 80% were achieved for carbonic anhydrase (29 kDa), 50% for aldolase (39 kDa), 46% for enolase (46 kDa), and 27% for glutamate dehydrogenase (56 kDa), and up to 74% sequence coverage was obtained for 25 kDa antibody drug conjugate subunits in online LC-MS experiments.

LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY BOTTOM‐UP PROTEOMIC METHODS IN ANIMAL SPECIES ANALYSIS OF PROCESSED MEAT FOR FOOD AUTHENTICATION AND THE DETECTION OF ADULTERATIONS

This review offers an overview of the current status and the most recent advances in liquid chromatography-mass spectrometry (LC-MS) techniques with both high-resolution and low-resolution tandem mass analyzers applied to the identification and detection of heat-stable species-specific peptide markers of meat in highly processed food products. We present sets of myofibrillar and sarcoplasmic proteins, which turned out to be the source of 105 heat-stable peptides, detectable in processed meat using LC-MS/MS. A list of heat-stable species-specific peptides was compiled for eleven types of white and red meat including chicken, duck, goose, turkey, pork, beef, lamb, rabbit, buffalo, deer, and horse meat, which can be used as markers for meat authentication. Among the 105 peptides, 57 were verified by multiple reaction monitoring, enabling identification of each species with high specificity and selectivity. The most described and monitored species by LC-MS/MS so far are chicken and pork with 26 confirmed heat-stable peptide markers for each meat. In thermally processed samples, myosin, myoglobin, hemoglobin, l-lactase dehydrogenase A and β-enolase are the main protein sources of heat-stable markers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Two New Lignan Derivatives from Jatropha curcas

Two new lignan derivatives, jatrolignan A (1) and B (2), were isolated from the aerial parts of Jatropha curcas. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR experiments, as well as high-resolution ESI mass analysis.

Bioanalytical and Mass Spectrometric Methods for Aldehyde Profiling in Biological Fluids.

Human exposure to aldehydes is implicated in multiple diseases including diabetes, cardiovascular diseases, neurodegenerative disorders (i.e., Alzheimer’s and Parkinson’s Diseases), and cancer. Because these compounds are strong electrophiles, they can react with nucleophilic sites in DNA and proteins to form reversible and irreversible modifications. These modifications, if not eliminated or repaired, can lead to alteration in cellular homeostasis, cell death and ultimately contribute to disease pathogenesis. This review provides an overview of the current knowledge of the methods and applications of aldehyde exposure measurements, with a particular focus on bioanalytical and mass spectrometric techniques, including recent advances in mass spectrometry (MS)-based profiling methods for identifying potential biomarkers of aldehyde exposure. We discuss the various derivatization reagents used to capture small polar aldehydes and methods to quantify these compounds in biological matrices. In addition, we present emerging mass spectrometry-based methods, which use high-resolution accurate mass (HR/AM) analysis for characterizing carbonyl compounds and their potential applications in molecular epidemiology studies. With the availability of diverse bioanalytical methods presented here including simple and rapid techniques allowing remote monitoring of aldehydes, real-time imaging of aldehydic load in cells, advances in MS instrumentation, high performance chromatographic separation, and improved bioinformatics tools, the data acquired enable increased sensitivity for identifying specific aldehydes and new biomarkers of aldehyde exposure. Finally, the combination of these techniques with exciting new methods for single cell analysis provides the potential for detection and profiling of aldehydes at a cellular level, opening up the opportunity to minutely dissect their roles and biological consequences in cellular metabolism and diseases pathogenesis.

Reconsidering mass spectrometric fragmentation in electron ionization mass spectrometry - new insights from recent instrumentation and isotopic labelling exemplified by ketoprofen and related compounds.

In various fields of chemical analyses, structurally unknown analytes are considered. Proper structure confirmation may be challenged by the low amounts of analytes that are available, e.g. in early stage drug development, in metabolism studies, in toxicology or in environmental analyses. In these cases, mass spectrometric techniques are often used to build up structure proposals for these unknowns. Fragmentation reactions in mass spectrometry are known to follow definite pathways that may help to assign structural elements by fragment ion recognition. This work illustrates an investigation of fragmentation reactions for gas chromatography/electron ionization mass spectrometric characterization of benzophenone derivatives using the analgesic drug ketoprofen and seven of its related compounds as model compounds. Deuteration and 18 O-labelling experiments along with high-resolution accurate mass and tandem mass spectrometry (MS/MS) were used to further elucidate fragmentation pathways and to substantiate rationales for structure assignments. Low-energy ionization was investigated to increase confidence in the identity of the molecular ion. The high-resolution mass analyses yielded unexpected differences that led to reconsideration of the proposals. Site-specific isotopic labelling helped to directly trace back fragment ions to their respective structural elements. The proposed fragmentation pathways were substantiated by MS/MS experiments. The described method may offer a perspective to increase the level of confidence in unknown analyses, where reference material is not (yet) available.

Identification of new process-related impurity in the key intermediate in the synthesis of TCV-116.

Development of safe and effective drugs requires complete impurity evaluation and, therefore, knowledge about the formation and elimination of impurities is necessary. During impurity profiling of a key intermediate during synthesis of candesartan cilexetil (1-(((cyclohexyloxy)carbonyl) oxy)ethyl 1-((2'-(2H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl) methyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxylate, TCV-116), a novel compound, which had not been reported previously, was observed. Structural elucidation of impurity was achieved by liquid chromatography hyphenated to different high resolution mass analyzers. Based on exact mass measurements and fragmentation pattern, a chloro alkyl carbonate ester analogue of the intermediate was identified. Structure of the impurity was confirmed by mass spectro-metric and NMR analyses of the target substance. Identified impurity could represent a hazard if it is transferred to the final API stage and its presence should be kept below allowed limits. Further investigation could reveal whether bis(1-chloroethyl) carbonate is a precursor to impurity formation. Therefore, synthesis should be regulated so as to minimize impurity production. Analysis of the final product indicated that the amount of impurity did not exceed 50 mg L-1, which represents the detection limit, determined according to the signal/noise ratio.

A novel strategy for rapid screening of the complex triterpene saponin mixture present in the methanolic extract of blackberry leaves (Rubus cv. Loch Ness) by UHPLC/QTOF-MS

Triterpene saponins are important chemical constituents in blackberry leaves and have significant antimicrobial activity, among other healthful properties.In this study, a new UHPLC-MS method was optimized with outstanding efficiency for a non-targeted metabolic approach and comprehensive analysis of bioactive compounds in blackberry leaves (Rubus cv. Loch Ness). With minimum sample treatment, phenols and triterpene saponins, among others, were separated according to their elution times. Once separated, the major triterpene saponins were classified by their aglycone moieties and their structures tentatively identified based on their accurate mass spectra in positive and negative ESI mode. By the use of UHPLC coupled to a TOF, a high-resolution and accuracy mass analyzer, reliable molecular formulas of the detected compounds were predicted, and along with the generated MS spectra in full scan mode, allowed the tentative identification and classification of the most abundant triterpene saponins presented the samples analyzed.A rapid and comprehensive strategy to study the complex saponin profile is presented. Unlike other LC–MS/MS methods, the proposed method requires just one mass analyzer, and to our knowledge, this is the first systematic study on triterpene saponins in blackberry leaves.

Aqueous chlorination of benzodiazepines diazepam and oxazepam: Kinetics, transformation products and reaction pathways

The pharmaceutical benzodiazepine diazepam and its metabolite oxazepam have been detected in wastewater treatment plants effluents, surface water and treated drinking water, but their transformation during chlorination process is not well understood. We investigated the reactions of diazepam and oxazepam with free available chlorine using a high-resolution mass analyzer and theoretical calculation to elucidate the fate of benzodiazepines during water chlorination process. The obtained apparent second-order rate constants (kapp) for chlorine reaction with diazepam and oxazepam varied from 0.01 to 1.7 M−1 s−1 and 0.7 to 31.6 M−1 s−1 in the pH range of 5.5–10.0, respectively. Under typical wastewater disinfection conditions of neutral pH values, free chlorine concentrations of 5 mg L−1 and contact times of up to 2 h, the corresponding half-lives for diazepam (∼180 min) and oxazepam (∼61 min) suggest that diazepam will be partly transformed during disinfection. Conversely, oxazepam will be considerably transformed during wastewater disinfection. The pH-dependency of kapp for diazepam could be explained by the reactions between neutral diazepam and HClO species. The kinetic pattern for oxazepam can be well described by species-specific reactions involving oxazepam or Cl2 and Cl2O species. In total, fifteen and eight transformation products were identified for chlorination of diazepam and oxazepam, respectively. The C-3 of 1,4-benzodiazepine structure was the main site of attack, leading predominantly to the oxidation and then cleavage of the C(3)-N(4) bond, as well as diazepine ring contractions. Based on mass balance estimation, the main chlorination product for diazepam and oxazepam are 7-chloro-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2,3-dione and 6-chloro-4-phenyl-2(1H)-quinazolinone, respectively.

Stimulated Motion Suppression (STMS): a New Approach to Break the Resolution Barrier for Ion Trap Mass Spectrometry.

Ion trap is an excellent platform to perform tandem mass spectrometry (MS/MS), but has an intrinsic drawback in resolving power. Using ion resonant ejection as an example, the resolution degradation can be largely attributed to the broadening of the resonant frequency band (RFB) between ion motion and driving alternative-current (AC). To solve this problem, stimulated motion suppression (STMS) was developed. The key idea of STMS is the use of two suppression alternative-current (SAC) signals, which both have reversed initial phases to the main AC. The SACs can block the unexpected sideband ion resonances (or ejections), therefore playing a key role in sharpening the RFB. The proof-of-concept has been demonstrated through ion trajectory simulations and validated experimentally. STMS provides a new and versatile means for the improvement of the ion trap resolution, which for a long time has reached the bottleneck through conventional methods, e.g., increasing the radio-frequency (RF) voltage and decreasing the mass scan rate. At the end, it is worth noting that the idea of STMS is very general and principally can be applied in any RF device for the purposes of high-resolution mass analysis and ion isolation. Graphical Abstract ᅟ.