Generation of substructure identification rules using feature-combinations from tandem mass spectra

Software to interpret tandem mass spectra, entitled Method for Analyzing Patterns in Spectra (MAPS), has been developed to provide substructure information for an automated compound identification system, This software consists of several program modules which manipulate databases of tandem mass spectra and substructure information, generate substructure identification rules, and apply these rules to the tandem mass spectra of unknown compounds to identify components of their structure. The MAPS rule generation program has been modified to generate rules based on specific combinations of spectral features that occur concertedly. False positives are drastically reduced by searching for “feature-combinations” that have 100% uniqueness with respect to a reference database of compounds. Recall is increased by the determination of multiple feature-combinations indicative of the presence of a given substructure. Strategies were developed in the algorithm for the discovery of feature-combinations that avoid the computation “explosion” that occurs when working with a large number of spectral features. The rules developed have the form: “IF feature-eombination a (FC a) or FC b,..., or FC x, THEN substructure SSn is present.”     

Optimization of automatically generated rules for predicting the presence and absence of substructures from MS and MS/MS data

A pattern-recognition/artificial-intelligence program, referred to as MAPS (Method for Analyzing Patterns in Spectra), was recently developed to identify the relationships that exist between substructures and the characteristic features they produce in the spectra from mass spectrometry (MS) and successive mass spectrometry (MS/MS). MAPS has been extended to utilize these relationships to formulate exclusion rules as well as inclusion rules, so that the absence of recognized substructures can be predicted as well as their presence. The potential usefulness of each MS and MS/MS spectral feature in such rule formulation is characterized by correlation and uniqueness factors. The correlation factor expresses the degree of correlation between a feature and a specific substructure; the uniqueness factor expresses the uniqueness of a feature with respect to that substructure. Features with high correlation factors are most use for predicting the absence of substructures, whereas features with high uniqueness factors are most useful for predicting their presence. Feature intensity-data have been found to improve the inclusion-rule performance and degrade the exclusion-rule performance. Criteria for optimizing the predictive abilities of both rule types are discussed.     

On the inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library: 1. Results of an Austrian multicenter study

The inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole‐quadrupole‐time‐of‐flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision‐energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole‐quadrupole‐time‐of‐flight, quadrupole‐quadrupole‐linear ion trap, quadrupole‐quadrupole‐quadrupole, and linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter‐instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent. Copyright © 2009 John Wiley & Sons, Ltd.     

Multidimensional computer evaluation of mass spectra

The increasing importance of spectroscopic methods as an analytical tool in industry, combined with the trend to automatize spectrometers, demands new standards in the quantity and quality of spectrum interpretation. Suitable computer programs should be able to predict structural features from mass spectral properties. The knowledge base is a structure-oriented mass spectral data collection consisting of some 42000 spectra and topologies. The comparison of selected mass spectral properties such as similarity, neutral losses and ion series of the unknown with the equivalent properties of the library spectra results in a set of corresponding structures. Subsequent substructure analysis yields a histogram of substructure frequencies containing information about their statistical relevance. The relevant substructure set may be recombined to produce a structure proposal, as is demonstrated for 1-acetyl-2-methoxy-4-trimethylsilyioxybenzene. In a second example, the relevant substructures derived by the interpretation system are used as input for the ¹³C-NMR substructure generator. This procedure reduces the solution space of the structure prediction algorithm considerably. Besides the spectrum interpretation, additional possibilities are available. The substructure search enables us, for example, to look for mass spectrometric reaction centres. Beyond that, substructure analysis is applicable to the determination of structural features typical of certain combinations of neutral losses and/or characteristic fragments.     

SANIST: optimization of a technology for compound identification based on the European Union directive with applications in forensic,pharmaceutical and food analyses

Electrospray Ionization and collision induced dissociation tandem mass spectrometry are usually employed to obtain compound identification through a mass spectra match. Different algorithms have been developed for this purpose (for example the nist match algorithm). These approaches compare the tandem mass spectra of the unknown analyte with the tandem mass spectra spectra of known compounds inserted in a database. The compounds are usually identified on the basis of spectral match value associated with a probability of recognition. However, this approach is not usually applied to multiple reaction monitoring transition spectra achieved by means of triple quadrupole apparatus, mainly due to the lack of a transition spectra database. The Surface Activated Chemical Ionization‐Electrospray‐NIST Bayesian model database search (SANIST) platform has been recently developed for new potential metabolite biomarker discovery, to confirm their identity and to use them for clinical and diagnostic applications. Here, we present an improved version of the SANIST platform that extends its application to forensic, pharmaceutical, and food analysis studies, where the compound identification rules are strict. The European Union (EU) has set directives for compound identification (EU directive 2002/657/EC). We have applied the SANIST method to identification of 11‐nor‐9‐carboxytetrahydro‐cannabinol in urine samples (an example of a forensic application), circulating levels of the immunosuppressive drug tacrolimus in blood (an example of a pharmaceutical application) and glyphosate in fruit juice (an example of a food analysis application) that meet the EU directive requirements. Copyright © 2016 John Wiley & Sons, Ltd.     

Chemical substructure identification by mass spectral library searching

A library-search procedure that identifies structural features of an unknown compound from its electron-ionization mass spectrum is described. Like other methods, this procedure first retrieves library compounds whose spectra are most similar to the spectrum of an unknown compound. It then deduces structural features of the unknown compound from the chemical structures of the retrievals. Unlike other methods, the significance of each retrieved spectrum is weighted according to its similarity to the spectrum of the unknown compound. Also, a “peaks-in-common” screening step serves to reduce search times and an optimized dot product function provides the match factor. If the molecular weight of the unknown compound is provided, the identification of certain substructures can be improved by including “neutral loss” peaks. Correlations between the presence of a substructure in a test compound and its presence among library retrievals were derived from the results of searching the NIST/EPA/NIH reference library with a 7891 compound test set. These correlations allow the estimation of probabilities of substructure occurrence and absence in an unknown compound from the results of a library search. This method may be viewed as an optimization of the “K-nearest neighbor” method of Isenhour and co-workers, with improvements that arise from spectrum screening, peak scaling, an optimal distance measure, a relative-distance weighting scheme, and a larger reference library.     

Towards a full reference library of MS(n) spectra. Testing of a library containing 3126 MS2 spectra of 1743 compounds

A library consisting of 3766 MS(n) spectra of 1743 compounds, including 3126 MS2 spectra acquired mainly using ion trap (IT) and triple-quadrupole (QqQ) instruments, was composed of numerous collections/sources. Ionization techniques were mainly electrospray ionization and also atmospheric pressure chemical ionization and chemical ionization. The library was tested for the performance in identification of unknowns, and in this context this work is believed to be the largest of all known tests of product-ion mass spectral libraries. The MS2 spectra of the same compounds from different collections were in turn divided into spectra of 'unknown' and reference compounds. For each particular compound, library searches were performed resulting in selection by taking into account the best matches for each spectral collection/source. Within each collection/source, replicate MS2 spectra differed in the collision energy used. Overall, there were up to 950 search results giving the best match factors and their ranks in corresponding hit lists. In general, the correct answers were obtained as the 1st rank in up to 60% of the search results when retrieved with (on average) 2.2 'unknown' and 6.2 reference replicates per compound. With two or more replicates of both 'unknown' and reference spectra (the average numbers of replicates were 4.0 and 7.8, respectively), the fraction of correct answers in the 1st rank increased to 77%. This value is close to the performance of established electron ionization mass spectra libraries (up to 79%) found by other workers. The hypothesis that MS2 spectra better match reference spectra acquired using the same type of tandem mass spectrometer (IT or QqQ) was neither strongly proved nor rejected here. The present work shows that MS2 spectral libraries containing sufficiently numerous different entries for each compound are sufficiently efficient for identification of unknowns and suitable for use with different tandem mass spectrometers.     

On the inter‐instrument and the inter‐laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem‐in‐space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem‐in‐time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time‐of‐flight (TOF), quadrupole–hexapole–TOF, linear ion trap (LIT), 3‐D ion trap and LIT–Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with ‘relative average match probability’ >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of the Major Components of Resina Draconis Extract and Their Metabolites in Rat Urine by LC-ESI-MS n

The aim of our study was to employ a liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS ⁿ ) method for the identification of the major components of Resina Draconis extract (RDE) and their metabolites in rat urine. Based on the above, 18 compounds were tentatively identified from the RDE. Among them, 4 compounds were unambiguously characterized by the comparison of the retention time and mass spectra with those of reference compounds and 14 compounds were tentatively identified by comparing the mass spectra with those of literature. In vivo, 21 compounds, including 13 parent compounds and 8 metabolites, were detected in rat urine after oral administration of RDE. The results may be helpful for future research on traditional Chinese medicine.     

Identification of phenolic compounds in artichoke waste by high-performance liquid chromatography-tandem mass spectrometry

A new fast and efficient method combining liquid chromatography coupled to ionspray mass spectrometry in tandem mode with negative ion detection is described for the qualitative analysis of artichoke waste. Forty-five phenolic compounds were identified on the basis of their mass spectra in full scan mode, mass spectra in different MS-MS modes, and retention times compared with those of available reference substances. The major compounds were found to be both caffeoylquinic and dicaffeoylquinic acids, luteolin glucuronide, luteolin galactoside, quercetin, and some quercetin glycosides.     

Comparison of different signal thresholds on data dependent sampling in orbitrap and LTQ mass spectrometry for the identification of peptides and proteins in complex mixtures

We evaluate the effect of ion-abundance threshold settings for data-dependent acquisition on a hybrid LTQ-Orbitrap mass spectrometer, analyzing features such as the total number of spectra collected, the signal to noise ratio of the full MS scans, the spectral quality of the tandem mass spectra acquired, and the number of peptides and proteins identified from a complex mixture. We find that increasing the threshold for data-dependent acquisition generally decreases the quantity but increases the quality of the spectra acquired. This is especially true when the threshold setting is set above the noise level of the full MS scan. We compare two distinct experimental configurations: one where full MS scans are acquired in the Orbitrap analyzer while tandem MS scans are acquired in the LTQ analyzer, and one where both full MS and tandem MS scans are acquired in the LTQ analyzer. We examine the number of spectra, peptides, and proteins identified under various threshold conditions, and we find that the optimal threshold setting is at or below the respective noise level of the instrument regardless of whether the full MS scan is performed in the Orbitrap or in the LTQ analyzer. When comparing the high-throughput identification performance of the two analyzers, we conclude that, used at optimal threshold levels, the LTQ and the Orbitrap identify similar numbers of peptides and proteins. The higher scan speed of the LTQ, which results in more spectra being collected, is roughly compensated by the higher mass accuracy of the Orbitrap, which results in improved database searching and peptide validation software performance.     

Characterization of fifty-one flavonoids in a Chinese herbal prescription Longdan Xiegan Decoction by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and photodiode array detection

High-performance liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry and photodiode array detection (HPLC-DAD-ESI-MS(n)) was developed to identify and characterize the flavonoids in a Chinese formulated preparation, Longdan Xiegan Decoction (LXD). In total, fifty-one flavonoids (27 flavones, 10 flavanones, 7 chalcones, 5 flavonols and 2 isoflavones) were characterized. Eighteen compounds among them including a newly detected flavonoid, naringin, from the ingredient herbs, were unambiguously determined by comparing the retention times (t(R)), UV spectral data and mass fragmentation behaviors with those of the reference compounds. Another thirty-three compounds were tentatively identified by referencing to the reported data of their UV and MS spectra. The ESI-MS/MS fragmentation behavior of flavones (OMe-substituted, O-glycosides, C-glycosides), chalcones, flavonols and their appropriate characteristic pathways were proposed. In negative ion ESI-MS all the flavonoids yielded prominent [M--H](-) ions in the first order mass spectra. Fragmentation with a loss of mass of 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)), 56 Da (2CO) and the residues of glucose and glucuronic acid observed in the MS/MS spectra were useful for aiding the structural identification of the flavonoids investigated.     

Quality evaluation of tandem mass spectral libraries

Tandem mass spectral libraries are gaining more and more importance for the identification of unknowns in different fields of research, including metabolomics, forensics, toxicology, and environmental analysis. Particularly, the recent invention of reliable, robust, and transferable libraries has increased the general acceptance of these tools. Herein, we report on results obtained from thorough evaluation of the match reliabilities of two tandem mass spectral libraries: the MSforID library established by the Oberacher group in Innsbruck and the Weinmann library established by the Weinmann group in Freiburg. Three different experiments were performed: (1) Spectra of the libraries were searched against their corresponding library after excluding either this single compound-specific spectrum or all compound-specific spectra prior to searching; (2) the libraries were searched against each other using either library as reference set or sample set; (3) spectra acquired on different mass spectrometric instruments were matched to both libraries. Almost 13,000 tandem mass spectra were included in this study. The MSforID search algorithm was used for spectral matching. Statistical evaluation of the library search results revealed that principally both libraries enable the sensitive and specific identification of compounds. Due to higher mass accuracy of the QqTOF compared with the QTrap instrument, matches to the MSforID library were more reliable when comparing spectra with both libraries. Furthermore, only the MSforID library was shown to be efficiently transferable to different kinds of tandem mass spectrometers, including “tandem-in-time” instruments; this is due to the coverage of a large range of different collision energy settings—including the very low range—which is an outstanding characteristics of the MSforID library.     

Similarity search for a 1H-NMR spectroscopic data base

A similarity-search system is described for proton-NMR spectroscopy. In order to achieve fast retrieval of reference compounds, the ¹H-NMR spectra of the data base and of the unknown are encoded in a bitsring. The individual bits of the binary signature describe different features of the spectra. Part of the coupling information is coded in such a way that effects of magnetic field strength are taken into account. The encoding thus permits a fast search for identical and structurally similar reference compounds in the data base even when the spectra were recorded at different magnetic field strengths. Because the search consists of weighted comparison of bits, each of them describing different spectral features, a choice of different kinds of searches is possible with the same signature by selecting appropriate weight vectors. Thus specific spectroscopic features can be selected for the search. Such a context-sensitive similarity-search system allows, for example, a search for compounds having similar multiplicities or similar subspectra in a given (e.g., aromatic) region of the spectrum. Furthermore, by adjusting two “software knobs” which influence the normalization of the search results, the user can choose between the two extremes of forward and reverse search, and between an identity search, similarity search or classification search. The results were tested on a small library containing 550 spectra including some mixtures and duplicates recorded under different experimental conditions at 250 and 400 MHz.     

MetFusion: integration of compound identification strategies

Mass spectrometry (MS) is an important analytical technique for the detection and identification of small compounds. The main bottleneck in the interpretation of metabolite profiling or screening experiments is the identification of unknown compounds from tandem mass spectra. Spectral libraries for tandem MS, such as MassBank or NIST, contain reference spectra for many compounds, but their limited chemical coverage reduces the chance for a correct and reliable identification of unknown spectra outside the database domain. On the other hand, compound databases like PubChem or ChemSpider have a much larger coverage of the chemical space, but they cannot be queried with spectral information directly. Recently, computational mass spectrometry methods and in silico fragmentation prediction allow users to search such databases of chemical structures. We present a new strategy called MetFusion to combine identification results from several resources, in particular, from the in silico fragmenter MetFrag with the spectral library MassBank to improve compound identification. We evaluate the performance on a set of 1062 spectra and achieve an improved ranking of the correct compound from rank 28 using MetFrag alone, to rank 7 with MetFusion, even if the correct compound and similar compounds are absent from the spectral library. On the basis of the evaluation, we extrapolate the performance of MetFusion to the KEGG compound database. Copyright © 2013 John Wiley & Sons, Ltd.     

Computer-aided elucidation of structures by carbon-13 nuclear magnetic resonance The DARC-EPIOS Method: Characterization of Ordered Substructures by Correlating the Chemical Shifts of Their Bonded Carbon Atoms

A reference substructure (SS) for which all spectral information (I) associated with each carbon atom is expressed as a function of the molecular environment is proposed. This information is expressed by topological relations, within this substructure, for which the spectral responses of its “bonded atoms” are linearly related to external alkyl effects. A correlation space (SS/I) is built with these topological relations (1–4 per carbon substructure). The resolving capacity of this SS/I space is greatly enhanced by introducing, in addition to all the reference substructures, some functions such as heteroatoms, cyclic structures, and stereochemical situations. The action of these functions is estimated by a statistical study of a large population of compounds from the DARC-PLURIDATA ¹³C-n.m.r. bank. This work introduces the notion of multiatom-centered substructure/multi-chemical shift relations, the discriminating ability of which compares very favorably to that of the notion of atom-centered substructure/monochemical shift relations. The generic reference subtructure defined as ELCO_b with its multichemical shifts constitutes a simple structural whole which is useful for critical improvement of the experimental reference data (distribution of spectral information and medium effects) and has proved to be excellent for the elucidation of new structures.     

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.     

Towards a full reference library of MSn spectra. II: A perspective from the library of pesticide spectra extracted from the literature/Internet

To gain perspective on building full transferable libraries of MSⁿ spectra from their diverse/numerous collections, a new library was built from 1723 MS^>1 spectra (mainly MS² spectra) of 490 pesticides and related compounds. Spectra acquired on different types of tandem instruments in various experimental conditions were extracted from 168 literature articles and Internet sites. Testing of the library was based on searches where 'unknown' and reference spectra originated from different sources (mainly from different laboratories) were cross‐compared. The NIST 05 MS² library was added to the reference spectra. The library searches were performed with all the test spectra or were divided into different subsamples containing (a) various numbers of replicate spectra of test compounds or (b) spectra acquired from different instrument types. Thus, the dependence of true/false search (identification) result rates on different factors was explored. The percentage of 1^st rank correct identifications (true positives) for the only 'unknown' mass spectrum and two and more reference spectra and matching precursor ion m/z values was 89%. For qualified matches, above the cut‐off match factor, that rate decreased to 80%. The corresponding rates based on the best match for two and more 'unknown' and reference spectral replicates were 89–94%. For quadrupole instruments, the rates were even higher: 91–95% (one 'unknown' spectrum) and 90–100% (two and more such spectra). This study shows that MS² spectral libraries generated from the numerous literature/Internet sources are not less efficient for the goal of identification of unknown compounds including pesticides than very common EI‐MS¹ libraries and are almost as efficient as the most productive from current MS² spectral databases. Such libraries may be used as individual reference databases or supplements to large experimental spectral collections covering many groups of abundant compounds and different types of tandem mass spectrometers. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapid screening and identification of cytochalasins by electrospray tandem mass spectrometry

The cytochalasin class of fungal metabolites was analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in microbial extracts. ESI-MS analyses of reference cytochalasins were performed and several product ions were produced in MS/MS experiments on parent ions that are structurally characteristic. A precursor ion search was performed to detect cytochalasins in an ethyl acetate extract of fungal strain RK97-F21. Three cytochalasins were detected and one of the components was identified as epoxycytochalasin H by comparing the tandem mass spectra of the product ions with those of reference compounds. This finding was further validated by LC/MS and LC/MS/MS experiments.