Identification Of Complex Mixtures Research Articles

The Molecules Gateway: A Homogeneous, Searchable Database of 150k Annotated Molecules from Actinomycetes.

Natural products are a sustainable resource for drug discovery, but their identification in complex mixtures remains a daunting task. We present an automated pipeline that compares, harmonizes and ranks the annotations of LC-HRMS data by different tools. When applied to 7,400 extracts derived from 6,566 strains belonging to 86 actinomycete genera, it yielded 150,000 molecules after processing over 50 million MS features. The web-based Molecules Gateway provides a highly interactive access to experimental and calculated data for these molecules, along with the metadata related to extracts and producer strains. We show how the Molecules Gateway can be used to rapidly identify known hard to find microbial products, unreported analogs of known families and not yet described metabolites. The Molecules Gateway, which complements available repositories, contains annotated MS data, both acquired and computationally processed under an identical workflow, making it suitable for global analyses which reveal a large and untapped chemical diversity afforded by actinomycetes.

On the part that NMR should play in mass spectrometry metabolomics in natural products studies

The field of metabolomics has witnessed remarkable growth in the context of natural products studies, with Mass Spectrometry (MS) being the predominant analytical tool for data acquisition. However, MS has inherent limitations when it comes to the structural elucidation of key metabolites, which can hinder comprehensive compound identification. This review paper discusses the integration of Nuclear Magnetic Resonance (NMR) spectroscopy as a complementary technique to address these limitations. We explore the concept of Quality Control (QC) samples, emphasizing their potential use for in-depth compound annotation and identification. Additionally, we discuss NMR’s advantages, limitations, and strategies to enhance sensitivity. We present examples where MS alone falls short in delivering accurate compound identification and introduce various tools for NMR compound identification in complex mixtures and the integration of MS and NMR data. Finally, we delve into the concept of DBsimilarity to broaden the chemical space understanding, aiding in compound annotation and the creation of compound lists for specific sample analyses.

Versa DB: Assisting 13C NMR and MS/MS Joint Data Annotation Through On‐Demand Databases

AbstractCompound identification in complex mixtures by NMR and MS is best achieved through experimental databases (DB) mining. Experimental DB frequently show limitations regarding their completeness, availability or data quality, thus making predicted database of increasing common use. Querying large databases may lead to select unlikely structure candidates. Two approaches to dereplication are thus possible: filtering of a large DB before search or scoring of the results after a large scale search. The present work relies on the former approach. As far as we know, nmrshiftdb2 is the only open‐source 13NMR chemical shift predictor that can be freely operated in batch mode. CFM‐ID 4.0 is one of the best‐performing open‐source tools for ESI‐MS/MS spectra prediction. LOTUS is a freely usable and comprehensive collection of secondary metabolites. Integrating the open source database and software LOTUS, CFM‐ID, and nmrshiftdb2 in a dereplication workflow requires presently programming skills, owing to the diversity of data encoding and processing procedures. A graphical user interface that integrates seamlessly chemical structure collection, spectral data prediction and database building still does not exist, as far as we know. The present work proposes a stand–alone software tool that assists the identification of mixture components in a simple way.

SMART-Miner: A convolutional neural network-based metabolite identification from 1 H-13 C HSQC spectra.

The identification of metabolites from complex biofluids and extracts of tissues is an essential process for understanding metabolic profiles. Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics studies for identification and quantification of metabolites. However, the accurate identification of individual metabolites is still a challenging process with higher peak intensity or similar chemical shifts from different metabolites. In this study, we applied a convolutional neural network (CNN) to 1 H-13 C HSQC NMR spectra to achieve accurate peak identification in complex mixtures. The results reveal that the neural network was successfully trained on metabolite identification from these 2D NMR spectra and achieved very good performance compared with other NMR-based metabolomic tools.

Algorithm-supported, mass and sequence diversity-oriented random peptide library design

Random peptide libraries that cover large search spaces are often used for the discovery of new binders, even when the target is unknown. To ensure an accurate population representation, there is a tendency to use large libraries. However, parameters such as the synthesis scale, the number of library members, the sequence deconvolution and peptide structure elucidation, are challenging when increasing the library size. To tackle these challenges, we propose an algorithm-supported approach to peptide library design based on molecular mass and amino acid diversity. The aim is to simplify the tedious permutation identification in complex mixtures, when mass spectrometry is used, by avoiding mass redundancy. For this purpose, we applied multi (two- and three-)-objective genetic algorithms to discriminate between library members based on defined parameters. The optimizations led to diverse random libraries by maximizing the number of amino acid permutations and minimizing the mass and/or sequence overlapping. The algorithm-suggested designs offer to the user a choice of appropriate compromise solutions depending on the experimental needs. This implies that diversity rather than library size is the key element when designing peptide libraries for the discovery of potential novel biologically active peptides.

Low-Cost and Simple Fabrication of Nanoplasmonic Paper for Coupled Chromatography Separation and Surface Enhanced Raman Detection.

Surface-enhanced Raman scattering (SERS) is a powerful analytical tool which enables the detection and identification of analytes adsorbed on nanostructured noble metals. However, SERS analysis of complex mixtures can be challenging due to spectral overlap and interference. In this report, we demonstrate a method to simplify the identification of mixed-analyte samples by coupling SERS detection with chromatographic separation on a nanoplasmonic paper substrate. This "nanopaper" substrate is a silver coated glass microfiber filter paper which possesses large SERS enhancement and can serve as a stationary phase in paper chromatography. Nanopaper is easily synthesized using the silver mirror reaction, making it a highly accessible technology. Nanopaper was successfully used as a combined paper chromatography-SERS (PC-SERS) substrate in the separation and identification of mixed organic dyes. It was further employed to separate and identify lycopene and β-carotene in commercial food products, demonstrating the versatility and utility of nanopaper in the identification of complex mixtures.

MS/MS-Free Protein Identification in Complex Mixtures Using Multiple Enzymes with Complementary Specificity

In this work, we present the results of evaluation of a workflow that employs a multienzyme digestion strategy for MS1-based protein identification in "shotgun" proteomic applications. In the proposed strategy, several cleavage reagents of different specificity were used for parallel digestion of the protein sample followed by MS1 and retention time (RT) based search. Proof of principle for the proposed strategy was performed using experimental data obtained for the annotated 48-protein standard. By using the developed approach, up to 90% of proteins from the standard were unambiguously identified. The approach was further applied to HeLa proteome data. For the sample of this complexity, the proposed MS1-only strategy determined correctly up to 34% of all proteins identified using standard MS/MS-based database search. It was also found that the results of MS1-only search were independent of the chromatographic gradient time in a wide range of gradients from 15-120 min. Potentially, rapid MS1-only proteome characterization can be an alternative or complementary to the MS/MS-based "shotgun" analyses in the studies, in which the experimental time is more important than the depth of the proteome coverage.

Challenges associated with the sampling and analysis of organosulfur compounds in air using real-time PTR-ToF-MS and offline GC-FID

Abstract. Organosulfur compounds (OSCs) are naturally emitted via various processes involving phytoplankton and algae in marine regions, from animal metabolism, and from biomass decomposition inland. These compounds are malodorant and reactive. Their oxidation to methanesulfonic and sulfuric acids leads to the formation and growth of atmospheric particles, which are known to influence clouds and climate, atmospheric chemical processes. In addition, particles in air have been linked to negative impacts on visibility and human health. Accurate measurements of the OSC precursors are thus essential to reduce uncertainties in their sources and contributions to particle formation in air. Two different approaches, proton-transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) and canister sampling coupled to gas chromatography with flame ionization detector (GC-FID), are compared for both laboratory standards (dimethyl sulfide, DMS; dimethyl disulfide, DMDS; dimethyl trisulfide, DMTS; and methanethiol, MTO) and for a complex sample. Results show that both techniques produce accurate quantification of DMS. While PTR-ToF-MS provides real-time measurements of all four OSCs individually, significant fragmentation of DMDS and DMTS occurs, which can complicate their identification in complex mixtures. Canister sampling coupled with GC-FID provides excellent sensitivity for DMS, DMDS, and DMTS. However, MTO was observed to react on metal surfaces to produce DMDS and, in the presence of hydrogen sulfide, even DMTS. Avoiding metal in sampling systems seems to be necessary for measuring all but dimethyl sulfide in air.

DART – LTQ ORBITRAP as an expedient tool for the identification of synthetic cannabinoids

Synthetic cannabinoids as designer drugs constitute a major problem due to their rapid increase in number and the difficulties connected with their identification in complex mixtures. DART (Direct Analysis in Real Time) has emerged as an advantageous tool for the direct and rapid analysis of complex samples by mass spectrometry. Here we report on the identification of six synthetic cannabinoids originating from seized material in various matrices, employing the combination of ambient pressure ion source DART and hybrid ion trap - LTQ ORBITRAP mass spectrometer. This report also describes the sampling techniques for the provided herbal material containing the cannabinoids, either directly as plant parts or as an extract in methanol and their influence on the outcome of the analysis. The high resolution mass spectra supplied by the LTQ ORBITRAP instrument allowed for an unambiguous assignment of target compounds. The utilized instrumental coupling proved to be a convenient way for the identification of synthetic cannabinoids in real-world samples.

Characterization of quinone derived protein adducts and their selective identification using redox cycling based chemiluminescence assay

The cytotoxic mechanism of many quinones has been correlated to covalent modification of cellular proteins. However, the identification of relevant proteins targets is essential but challenging goals. To better understand the quinones cytotoxic mechanism, human serum albumin (HSA) was incubated in vitro with different concentration of menadione (MQ). In this respect, the initial nucleophilic addition of proteins to quinone converts the conjugates to redox-cycling quinoproteins with altered conformation and secondary structure and extended life span than the short lived, free quinones. The conjugation of MQ with nucleophilic sites likewise, free cysteine as well as ɛ-amino group of lysine residue of HSA has been found to be in concentration dependent manner. The conventional methods for modified proteins identification in complex mixtures are complicated and time consuming. Herein, we describe a highly selective, sensitive, simple, and fast strategy for quinoproteins identification. The suggested strategy exploited the unique redox-cycling capability of quinoproteins in presence of a reductant, dithiothreitol (DTT), to generate reactive oxygen species (ROS) that gave sufficient chemiluminescence (CL) when mixed with luminol. The CL approach is highly selective and sensitive to detect the quinoproteins in ten-fold molar excess of native proteins without adduct enrichment. The approach was also coupled with gel filtration chromatography (GFC) and used to identify adducts in complex mixture of proteins in vitro as well as in rat plasma after MQ administration. Albumin was identified as the main protein in human and rat plasma forming adduct with MQ. Overall, the identification of quinoproteins will encourage further studies of toxicological impact of quinones on human health.

Rapid identification of coumarins from Micromelum falcatum by UPLC-HRMS/MS and targeted isolation of three new derivatives.

Micromelum falcatum, a medicinal plant of the Rutaceae family, has been used in the Traditional Chinese Medicine (TCM) mainly against colds and rheumatoid arthritis. Despite its traditional use the association of its constituents with possible anti-inflammatory activity has not been explored. During this study, a rapid UPLC-ESI(+)-HRMS method was developed for the profiling of M. falcatum leave extracts and the targeted isolation of coumarin constituents. Based on chromatographic, spectroscopic and spectrometric features several 7-oxygenated coumarin derivatives were detected. After targeted isolation, eight coumarins, among them three new natural products, namely microfalcrin, microcoumaririn and micromelosidester, were purified using semi-preparative HPLC and unambiguously identified by 1 and 2D NMR. Furthermore, important spectrometric characteristics were revealed based on the HRMS and HRMS/MS spectra of the isolated 7-oxygenated coumarins facilitating their identification in complex mixtures. Finally, the anti-inflammatory properties of the extracts and representative compounds were evaluated by measuring the inhibition of the pro-inflammatory mediator NF-κB induction and nitric oxide (NO) production.

Integrated Sample Preparation Methodology for Proteomics: Analysis of Native Proteins

An innovative sample preparation strategy is reported for protein identification in complex mixtures based on integration of affinity chromatographic selection and accelerated trypsin digestion using a continuous flow immobilized enzyme reactor (cf-IMER). Affinity selected glycoproteins were released to a cf-IMER column which converted native proteins to peptides in 5 min at elevated temperature. Digestion with the cf-IMER was compared to the traditional 16 h solution-based trypsin digestion of reduced and alkylated proteins. With immobilized antibody selection of Lewis x (Le(x)) glycan bearing glycoproteins from plasma, 66 proteins were identified in total with the two methods while approximately 1/3 of the total proteins and peptides were only observed with the cf-IMER. This suggests that proteomics based on protein identification by reduction and alkylation with solution-based trypsin digestion alone may not be identifying large numbers of proteins or peptides present at detectable levels in samples. Furthermore, except for proteins containing a high content of disulfide bonds, the majority of proteins did not require reduction and alkylation steps for their identification. The validity of the proposed proteolysis was evaluated in several ways by analyses of a model protein and yeast lysates where the reproducibility of quantification was essentially the same with both cf-IMER and solution-based proteolysis.

Linear Ion Trap MSn of Enzymatically Synthesized 13C-Labeled Fructans Revealing Differentiating Fragmentation Patterns of β (1-2) and β (1-6) Fructans and Providing a Tool for Oligosaccharide Identification in Complex Mixtures

Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of β-d-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as Lolium perenne. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MS(n) method for the separation and detection of fructan isomers and present here evidence for specific MS(n) fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MS(n) analysis of (13)C labeled fructan oligomers produced by L. perenne fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards.

CHIRPED-PULSE FOURIER TRANSFORM MICROWAVE SPECTROSCOPY: A NEW TECHNIQUE FOR RAPID IDENTIFICATION OF CHEMICAL AGENTS

We have developed a new broadband Chirped-Pulse Fourier Transform Microwave (CP-FTMW) spectrometer that allows the microwave spectrum in the 7.5-18.5 GHz range to be measured in a single data event. This technique produces a pure rotational spectrum that can be used for unambiguous identification of any species having a permanent electric dipole moment. CP-FTMW is a gas phase technique that is ideally suited for the detection of airborne chemical warfare agents (CWA) which must be detected in trace amounts (<10 ppm in air). The high resolution of the technique allows the identification of complex mixtures without the need for a preliminary separation step, such as gas chromatography, which significantly reduces analysis time. The technique is “blind” to major atmospheric components ( N 2, O 2, CO 2, H 2 O ) as they either do not posses a permanent dipole moment or do not absorb in the range of the spectrometer, thereby eliminating large background signals. In this paper we will present preliminary results that are focused on early detection of airborne CWA, including acquisition time, sensitivity limits, and sample handling requirements for several of these species.

Combination of HPLC “Inverted Chirality Columns Approach” and MS/MS Detection for Extreme Enantiomeric Excess Determination Even in Absence of Reference Samples. Application to Camptothecin Derivatives

An original, extremely sensitive and selective HPLC-MS/MS technique for the identification and determination of the minor enantiomer in nonracemic mixtures, even when only one enantiomer is available as reference, is described. The method is based on the so-called "inverted chirality columns approach" (ICCA) and consists of the use of chiral stationary phases (CSPs) available in both enantiomeric forms: in fact, inversion of the elution order for a pair of enantiomers is observed in response to the change in column chirality. This offers two key advantages: first, it is possible to demonstrate the potential enantioselectivity of the system by generating a virtual racemate, and second, it permits the choosing of the right column chirality for trace determination. Combination with MS/MS detection affords high specificity allowing not only high sensitivity (down to 0.0025% of the minor enantiomer) but also unequivocal peak identification in complex mixtures. Applications to semisynthetic derivatives of camptothecin, endowed with antitumor activity, are reported. Moreover, applicability of ICCA is not limited to this class of molecules but generates universal support. Its use might also be extended to other classes of compounds by using other CSPs, available in both enantiomeric forms.

Modification of Host Lipid Raft Proteome upon Hepatitis C Virus Replication

Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.

Protein Identification in Complex Mixtures

This paper investigates the prospects of successful mass spectrometric protein identification based on mass data from proteolytic digests of complex protein mixtures. Sets of proteolytic peptide masses representing various numbers of digested proteins in a mixture were generated in silico. In each set, different proteins were selected from a protein sequence collection and for each protein the sequence coverage was randomly selected within a particular regime (15-30% or 30-60%). We demonstrate that the Probity algorithm, which is characterized by an optimal tolerance for random interference, employed in an iterative procedure can correctly identify >95% of proteins at a desired significance level in mixtures composed of hundreds of yeast proteins under realistic mass spectrometric experimental constraints. By using a model of the distribution of protein abundance, we demonstrate that the very high efficiency of identification of protein mixtures that can be achieved by appropriate choices of informatics procedures is hampered by limitations of the mass spectrometric dynamic range. The results stress the desire to choose carefully experimental protocols for comprehensive proteome analysis, focusing on truly critical issues such as the dynamic range, which potentially limits the possibilities of identifying low abundance proteins.

Prediction of the gas chromatographic retention values of chlorobenzenes on different stationary phases by using structure–retention correlations

The retention values (capacity factors or retention indices) of the isomers of chlorobenzenes with two to six chlorine atoms were predicted by using structure–retention correlations and the calculated contributions of the ortho, meta and para positions of the substituent atoms on the aromatic ring. The method permits the prediction of the retention of every isomer by starting from the measured retention of a few compounds, and does not require the use of all of the possible isomers as standard samples for the identification in complex mixtures.

The application of chemical ionization mass spectrometry to the identification of urinary peptides

The use of chemical ionization (c.i.) mass spectrometry has been applied to the identification of complex mixtures of dipeptides, without the need for a prior separation of the components. The peptides are identified as their N-ethoxycarbonylpropenyl dimethyltrideuteromethyl ester derivatives. The technique was originally used by us for the identification of dipeptide mixtures from dipeptidylaminopeptidase-I digests of polypeptides, but was found to be a valuable procedure for the rapid identification of urinary peptides. The protocol of the procedure involves: (1) purification of the urinary peptides by column chromatography on G10 Sephadex followed by paper chromatography; (2) derivatization of the peptide fraction with ethylacetoacetate and dimethyltrideuteromethyl-phenylammonium hydroxide; (3) mass spectrometry (c.i.) of the derivatized fraction; (4) identification of the urinary dipeptides by computer matching of selected ions from the mass spectrum. This procedure was applied to the identification of dipeptides in the urine of a child suffering from chronic skin ulceration and oedema. At least 15 dipeptides were identified, most of which contained proline or hydroxyproline in the carboxyl terminal position. The results suggested that the patient suffered from a defect in collagen metabolism. This hypothesis was subsequently confirmed by a grossly diminished level of prolidase in cultured fibroblasts.

A New Approach to the Improved Separation and Identification of Complex Mixtures by Gas Chromatography I. Application of Naphtha

For the total analysis of a complex sample, like the naphtha, which contains a host of homologues and isomers in widely varying proportions, the efficiency of any single stationary liquid is definitely limited with respect to its power of fractionation, resolution and identification of the components. These deficiencies can be removed by using a pair of stationary liquids, one separating essentially according to vapour pressure and the other predominantly according to structural differentials of the solute molecules in both the stages of fractionation by preparative GC and subsequent resolution of the fractions by analytical GC. By choosing squalane and tetracyanoethylated pentaerythritol as the two stationary liquids, it has been demonstrated that the efficiency of the retention index and its extended concepts, like, ΔI, dI/dT etc., is vastly improved for the identification of hydrocarbon peaks and the interpretation of complex gas chromatograms. This principle can be easily extended to the qualitative and quantitative analysis of complex samples of all other chemical types, provided the choice of the pair of stationary liquids is proper and the desired retention data of the components are available in them.