A novel database search algorithm is presented for the qualitative identification of proteins over a wide dynamic range, both in simple and complex biological samples. The algorithm has been designed ...for the analysis of data originating from data independent acquisitions, whereby multiple precursor ions are fragmented simultaneously. Measurements used by the algorithm include retention time, ion intensities, charge state, and accurate masses on both precursor and product ions from LC-MS data. The search algorithm uses an iterative process whereby each iteration incrementally increases the selectivity, specificity, and sensitivity of the overall strategy. Increased specificity is obtained by utilizing a subset database search approach, whereby for each subsequent stage of the search, only those peptides from securely identified proteins are queried. Tentative peptide and protein identifications are ranked and scored by their relative correlation to a number of models of known and empirically derived physicochemical attributes of proteins and peptides. In addition, the algorithm utilizes decoy database techniques for automatically determining the false positive identification rates. The search algorithm has been tested by comparing the search results from a four-protein mixture, the same four-protein mixture spiked into a complex biological background, and a variety of other "system" type protein digest mixtures. The method was validated independently by data dependent methods, while concurrently relying on replication and selectivity. Comparisons were also performed with other commercially and publicly available peptide fragmentation search algorithms. The presented results demonstrate the ability to correctly identify peptides and proteins from data independent acquisition strategies with high sensitivity and specificity. They also illustrate a more comprehensive analysis of the samples studied; providing approximately 20% more protein identifications, compared to a more conventional data directed approach using the same identification criteria, with a concurrent increase in both sequence coverage and the number of modified peptides.
In the present work, an extensive study on the presence of eighteen pharmaceuticals and personal care products (PPCPs) in eight wastewater treatment plants (WWTPs) of Greece has been conducted. The ...study covered four sampling periods over 1-year, where samples (influents; effluents) from eight WWTPs of various cities in Greece were taken. All WWTPs investigated are equipped with conventional activated sludge treatment. A common pre-concentration step based on SPE was applied, followed by LC–UV/Vis–ESI–MS. Further confirmation of positive findings was accomplished by using LC coupled to a high resolution Orbitrap mass spectrometer. The results showed the occurrence of all target compounds in the wastewater samples with concentrations up to 96.65μg/L. Paracetamol, caffeine, trimethoprim, sulfamethoxazole, carbamazepine, diclofenac and salicylic acid were the dominant compounds, while tolfenamic acid, fenofibrate and simvastatin were the less frequently detected compounds with concentrations in effluents below the LOQ. The removal efficiencies showed that many WWTPs were unable to effectively remove most of the PPCPs investigated. Finally, the study provides an assessment of the environmental risk posed by their presence in wastewaters by means of the risk quotient (RQ). RQs were more than unity for various compounds in the effluents expressing possible threat for the aquatic environment. Triclosan was found to be the most critical compound in terms of contribution and environmental risk, concluding that it should be seriously considered as a candidate for regulatory monitoring and prioritization on a European scale on the basis of realistic PNECs. The results of the extensive monitoring study contributed to a better insight on PPCPs in Greece and their presence in influent and effluent wastewaters. Furthermore, the unequivocal identification of two transformation products of trimethoprim in real wastewaters by using the advantages of the LTQ Orbitrap capabilities provides information that should be taken into consideration in future PPCP monitoring studies in wastewaters.
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•Comprehensive study of 18 PPCPs in eight WWTPs in Greece•Further confirmation was accomplished by LC–MS/LTQ Orbitrap.•Report on the occurrence of trimethoprim transformation products in wastewaters•Occurrence of budesonide, a glucocorticoid steroid, is extensively studied in WWTPs.•Triclosan was the most critical compound in terms of contribution and environmental risk.
Pharmaceuticals require careful and precise determination of their impurities that might harm the user upon consumption. Although today, the most common technique for impurities identification is ...liquid chromatography‐mass spectrometry (LC‐MS/MS), it has several downsides due to the nature of the ionization method. Also, the analyses in many cases are targeted thus despite being present, some of the compounds will not be revealed. In this paper, we propose and show a new method for untargeted analysis and identification of impurities in active pharmaceutical ingredients (APIs). The instrument used for these analyses is a novel electron ionization (EI) LC‐MS with supersonic molecular beams (SMB). The EI‐LC‐MS‐SMB was implemented for analyses of several drug samples spiked with an impurity. The instrument provides EI mass spectra with enhanced molecular ions, named Cold EI, which increases the identification probabilities when the compound is identified with the aid of an EI library like National Institute of Standards and Technology (NIST). We analyzed ibuprofen and its impurities, and both the API and the expected impurity were identified with names and structures by the NIST library. Moreover, other unexpected impurities were found and identified proving the ability of the EI‐LC‐MS‐SMB system for truly untargeted analysis. The results show a broad dynamic range of four orders of magnitude at the same run with a signal‐to‐noise ratio of over 10 000 for the API and almost uniform response.
The detection, correlation, and comparison of peptide and product ions from a data independent LC-MS acquisition strategy with data dependent LC-MS/MS is described. The data independent mode of ...acquisition differs from an LC-MS/MS data acquisition since no ion transmission window is applied with the first mass analyzer prior to collision induced disassociation. Alternating the energy applied to the collision cell, between low and elevated energy, on a scan-to-scan basis, provides accurate mass precursor and associated product ion spectra from every ion above the LOD of the mass spectrometer. The method therefore provides a near 100% duty cycle, with an inherent increase in signal intensity due to the fact that both precursor and product ion data are collected on all isotopes of every charge-state across the entire chromatographic peak width. The correlation of product to precursor ions, after deconvolution, is achieved by using reconstructed retention time apices and chromatographic peak shapes. Presented are the results from the comparison of a simple four protein mixture, in the presence and absence of an enzymatically digested protein extract from Escherichia coli. The samples were run in triplicate by both data dependant analysis (DDA) LC-MS/MS and data-independent, alternate scanning LC-MS. The detection and identification of precursor and product ions from the combined DDA search results of the four protein mixture were used for comparison to all other data. Each individual set of data-independent LC-MS data provides a more comprehensive set of detected ions than the combined peptide identifications from the DDA LC-MS/MS experiments. In the presence of the complex E. coli background, over 90% of the monoisotopic masses from the combined LC-MS/MS identifications were detected at the appropriate retention time. Moreover, the fragmentation pattern and number of associated elevated energy product ions in each replicate experiment was found to be very similar to the DDA identifications. In the case of the corresponding individual DDA LC-MS/MS experiment, 43% of the possible detectable peptides of interest were identified. The presented data illustrates that the time-aligned data from data-independent alternate scanning LC-MS experiments is highly comparable to the data obtained via DDA. The obtained information can therefore be effectively and correctly deconvolved to correlate product ions with parent precursor ions. The ability to generate precursor-product ion tables from this information and subsequently identify the correct parent precursor peptide will be illustrated in a companion manuscript.
Summary
The term metabolome was coined in 1998, by analogy to genome, transcriptome and proteome. The first research papers using the terms metabolomics, metabonomics, metabolic profiling or ...metabolite profiling were published shortly thereafter. In this short review we reflect on the major achievements brought about by the use of these approaches, and document the knowledge and technology gaps that are currently constraining its further development. Finally, we detail why we think that the time is ripe to refocus our efforts on the understanding of metabolic function.
Significance Statement
In this short review we focus on the major achievements in metabolomics and document knowledge and technology gaps which are currently constraining its further development. In addition, we detail why we think that the time is ripe to refocus our efforts on the understanding of metabolic function.
In order to investigate the actual state of contamination of fisheries feeds by mycotoxins, we examined a method for simultaneously analyzing 10 species of trichothecenes in fisheries feeds. A ...pretreatment method with a high purification effect was constructed using a multifunctional column and graphite carbon, and a high-speed and highly robust analytical method was established by using a C18 column for UHPLC. When this analysis method was used to analyze commercially available fisheries feeds, several types of fungal toxins such as deoxynivalenol and its modified mycotoxins were detected in many feeds. All of the detected feeds contained wheat flour, suggesting the possibility of contamination of the raw material flour.
Fermentation is a critical manufacturing process to produce black tea, in which the chemical compositions are greatly changed. However, the dynamic changes of this sophisticated process are far from ...clear, and were often characterized by determining a small number of compounds. In this study, we applied a non-targeted metabolomics approach based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to comprehensively profile the variations of metabolites in tea samples with various fermentation durations of 0, 1, 2, 4, 6, 8, 10, 12 and 14h. Principal component analysis indicated obvious stepwise alterations of tea metabolome during the fermentation. Relative quantitation of 61 identified metabolites including catechins, dimeric catechins, flavonol glycosides, amino acids, phenolic acids, alkaloids, and nucleosides revealed distinct changes of phenol pathway. Dynamic changes of a part of these compounds were mapped for the first time. To the best of our knowledge, this study offered the most comprehensive profiles of metabolite changes during the tea fermentation process.
•A LC–MS based metabolomics approach was used to study the tea fermentation process.•Obvious stepwise alterations of the tea metabolome were observed.•Dynamic changes of 61 identified metabolites were profiled.•Changes of metabolites along the phenylpropanoid/flavonoid pathway were mapped.•A novel derivative of theanine was discovered and identified as theanine glucoside.
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