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.     

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.     

Combined use of ESI–QqTOF-MS and ESI–QqTOF-MS/MS with mass-spectral library search for qualitative analysis of drugs

The potential of the combined use of ESI–QqTOF-MS and ESI–QqTOF-MS/MS with mass-spectral library search for the identification of therapeutic and illicit drugs has been evaluated. Reserpine was used for standardizing experimental conditions and for characterization of the performance of the applied mass spectrometric system. Experiments revealed that because of the mass accuracy, the stability of calibration, and the reproducibility of fragmentation, the QqTOF mass spectrometer is an appropriate platform for establishment of a tandem-mass-spectral library. Three-hundred and nineteen substances were used as reference samples to build the spectral library. For each reference compound, product-ion spectra were acquired at ten different collision-energy values between 5 eV and 50 eV. For identification of unknown compounds, a library search algorithm was developed. The closeness of matching between a measured product-ion spectrum and a spectrum stored in the library was characterized by a value called “match probability”, which took into account the number of matched fragment ions, the number of fragment ions observed in the two spectra, and the sum of the intensity differences calculated for matching fragments. A large value for the match probability indicated a close match between the measured and the reference spectrum. A unique feature of the library search algorithm—an implemented spectral purification option—enables characterization of multi-contributor fragment-ion spectra. With the aid of this software feature, substances comprising only 1.0% of the total amount of binary mixtures were unequivocally assigned, in addition to the isobaric main contributors. The spectral library was successfully applied to the characterization of 39 forensic casework samples. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Computer identification of infrared spectra by correlation-based file searching

A new method for the computerized search and identification of infrared spectra has been developed and evaluated. Based on cross-correlation, the search system utilizes all spectral information in a digitized spectrum when it attempts to match an unknown spectrum to one in a small library of known spectra. To evaluate a spectral match, the search program calculates the cross-correlation function between the unknown and known (library) spectra which indicates their degree of similarity and allows library spectra to be ranked in order of probability of match to the unknown spectrum. In this study, several small infrared spectral libraries of structurally similar compounds were searched under conditions which examined the sensitivity of the search method to chemical and instrumental variations. Because the correlation technique is slower than conventional file-searching methods, it will probably find greatest use in the search of small collections of similar spectra or as a match-ranking procedure following preliminary selection by a faster search method.     

Evaluation of mass spectral library search algorithms implemented in commercial software

Performance of several library search algorithms (against EI mass spectral databases) implemented in commercial software products ( acd/specdb , chemstation , gc/ms solution and ms search ) was estimated. Test set contained 1000 mass spectra, which were randomly selected from NIST'08 (RepLib) mass spectral database. It was shown that composite (also known as identity) algorithm implemented in ms search (NIST) software gives statistically the best results: the correct compound occupied the first position in the list of possible candidates in 81% of cases; the correct compound was within the list of top ten candidates in 98% of cases. It was found that use of presearch option can lead to rejection of the correct answer from the list of possible candidates (therefore presearch option should not be used, if possible). Overall performance of library search algorithms was estimated using receiver operating characteristic curves. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Clipped representations of fourier-transform ion-cyclotron resonance mass spectra

Because of its high mass resolution over a wide mass range, a Fourier transform/mass spectrometry (F.t./m.s.) experiment may generate very large spectral data sets (? 64 K each), creating severe data storage problems for g.c./F.t./m.s. and for mass spectral library storage, search and retrieval. Fortunately, the stored experimental data need not be highly precise. In this paper, it is shown that useful mass spectra can be produced from time-domain F.t./m.s. data that have been clipped to only 1 bit/word, thereby offering a potential reduction by a factor of ? 20 in data storage requirement. In many cases, mass spectral resolution can actually be enhanced by the clipping process. Finally, it is shown tha a compound can be identified quickly and accurately by comparison of its clipped representation with similarly clipped data for library compounds.     

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.     

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.     

Testing an alternative search algorithm for compound identification with the ‘Wiley Registry of Tandem Mass Spectral Data,MSforID’

A tandem mass spectral database system consists of a library of reference spectra and a search program. State‐of‐the‐art search programs show a high tolerance for variability in compound‐specific fragmentation patterns produced by collision‐induced decomposition and enable sensitive and specific ‘identity search’. In this communication, performance characteristics of two search algorithms combined with the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’ (Wiley Registry MSMS, John Wiley and Sons, Hoboken, NJ, USA) were evaluated. The search algorithms tested were the MSMS search algorithm implemented in the NIST MS Search program 2.0g (NIST, Gaithersburg, MD, USA) and the MSforID algorithm (John Wiley and Sons, Hoboken, NJ, USA). Sample spectra were acquired on different instruments and, thus, covered a broad range of possible experimental conditions or were generated in silico. For each algorithm, more than 30 000 matches were performed. Statistical evaluation of the library search results revealed that principally both search algorithms can be combined with the Wiley Registry MSMS to create a reliable identification tool. It appears, however, that a higher degree of spectral similarity is necessary to obtain a correct match with the NIST MS Search program. This characteristic of the NIST MS Search program has a positive effect on specificity as it helps to avoid false positive matches (type I errors), but reduces sensitivity. Thus, particularly with sample spectra acquired on instruments differing in their setup from tandem‐in‐space type fragmentation, a comparably higher number of false negative matches (type II errors) were observed by searching the Wiley Registry MSMS. Copyright © 2013 John Wiley & Sons, Ltd.     

基于稀疏模型的气相色谱-质谱数据解析及其在严重重叠峰解析中的应用

提出一种气相色谱-质谱（GC-MS）数据解析算法。以色谱峰顶点处的质谱作为待测谱,在谱库中检索一定量相关参考谱,求解关于各纯组分色谱响应值的方程。质谱检索采取分步策略,先利用质谱碎片规律建立高速索引进行粗选,然后使用强峰高概率出峰准则和耐挤压性准则排除无关质谱。为求解色谱响应值方程,提出基于稀疏模型的回归算法,相比传统算法,稀疏算法利于提取待测谱的主要结构,避免"过拟合"。实验结果表明,该质谱检索算法具有较高的精度和规模较小的剩余参考谱集,而所提稀疏模型算法可有效解析严重重叠峰。该算法可作为GC-MS重叠峰解析,特别是严重重叠峰解析的一种有效解决方案。     

Comparison of different search engines using validated MS/MS test datasets

Massive amounts of tandem mass spectra are produced in high-throughput proteomics studies. The manual interpretation of these spectra is not feasible. Instead, search engines are used to match the tandem mass spectra with sequence information contained in proteomics and genomics databases. Typically, these search engines provide a list of the best matching peptide sequences for an individual tandem mass spectrum. As well, they provide scores that are somewhat related to the confidence level in the match. Many peptide tandem mass spectra search engines have been reported. These search engines provide very different results depending on the type of mass spectrometers used and their input parameters. Here we describe a comparative analysis of different search engines using validated test sets of tandem mass spectra. We have defined test sets of MS/MS spectra derived from high throughput proteomics experiments performed by HPLC-ESI-MS/MS on ion trap (LCQ) and tandem quadrupole time-of-flight instruments with a pulsar functionality (Qstar Pulsar) mass spectrometers. We analyzed the ability of the different search engines to identify the correct peptides, and the cross-validations of the different search engines.     

GC／MS检测信息的综合利用

从充分利用气相色谱／质谱（ＧＣ／ＭＳ）提供的信息来鉴定ＧＣ流出组分的目的出发，并以脂肪醇分析为例讨论了ＧＣ保留指数在ＭＳ谱图解析及ＭＳ鉴定结果的合理性检验方面的作用。总结了ＧＣ保留的同系物规律、极性规律、加和规律及异构物规律来指导ＭＳ谱图解析。提出了含氧化合物官能团相对于－ＣＨ２－质量数的保留指数附加贡献值辅助ＭＳ确定化合物的类别。同时采用了ＧＣ／ＭＳ中选择离子检测方法（ＳＩＭ）在分子离子峰受噪音干扰或太弱以至完全未观测到时来进一步佐证其相对分子质量。     

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.     

Use of PLS Discriminant Analysis for Revealing the Absence of a Compound in an Electron Ionization Mass Spectral Database

A mathematical model is proposed for revealing the absence of a compound to be identified in an electron impact mass spectral library. The mathematical model (developed based on PLS Discriminant Analysis) can be represented as a “black box” which provides an answer whether a compound to be sought is absent or present in a database. The match factors of top ten candidates among the possible ones were used as input data. More than 5000 objects (mass spectra) were used at the steps of training, validation, and testing. The developed classification model provides correct prediction (of whether a compound is absent from the library) in 28.4% cases, while only 1.2% of compounds present in the database were incorrectly classified as the absent ones.     

Evaluation of the comparability of spectra generated using a tuning point protocol on twelve electrospray ionisation tandem-in-space mass spectrometers

Product ion spectra produced by collision-induced dissociation (CID) in tandem mass spectrometry can yield important structural information on organic compounds which can aid in their identification. However, differences in experimental conditions may have a strong effect on the degree of product ion formation and therefore on the features observed in product ion spectra. For this reason, a common approach for library building is the acquisition of several spectra, typically between 5 and 10, each at a different collision energy level. In this study, the use of an alternative approach was investigated, where a tuning point protocol was applied to tune the instruments in an attempt to standardise CID conditions prior to data acquisition. With this approach, the acquisition of a single mass spectrum was sufficient. The stability of the tuning point was investigated and the choice of a commercially available search package to assess spectral comparability was discussed. Finally, the product ion spectra of 33 compounds were acquired on twelve tandem-in-space instruments, including nine triple quadrupoles, one hybrid triple quadrupole linear ion trap and two quadrupole time-of-flight mass spectrometers, resulting in 2178 spectral comparisons being carried out. The results from the spectral comparisons suggest that the use of a tuning point enables the standardisation of the experimental conditions that affect the degree of product ion formation. Indeed, 84.5% of the comparisons demonstrated a good degree of spectral agreement with match scores greater than 700, which we believe is the minimum score for a tentative library match.     

The use of MS classifiers and structure generation to assist in the identification of unknowns in effect-directed analysis

Structure generation and mass spectral classifiers have been incorporated into a new method to gain further information from low-resolution GC-MS spectra and subsequently assist in the identification of toxic compounds isolated using effect-directed fractionation. The method has been developed for the case where little analytical information other than the mass spectrum is available, common, for example, in effect-directed analysis (EDA), where further interpretation of the mass spectra is necessary to gain additional information about unknown peaks in the chromatogram. Structure generation from a molecular formula alone rapidly leads to enormous numbers of structures; hence reduction of these numbers is necessary to focus identification or confirmation efforts. The mass spectral classifiers and structure generation procedure in the program MOLGEN-MS was enhanced by including additional classifier information available from the NIST05 database and incorporation of post-generation ‘filtering criteria’. The presented method can reduce the number of possible structures matching a spectrum by several orders of magnitude, creating much more manageable data sets and increasing the chance of identification. Examples are presented to show how the method can be used to provide ‘lines of evidence’ for the identity of an unknown compound. This method is an alternative to library search of mass spectra and is especially valuable for unknowns where no clear library match is available.     

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.     

Unravelling the composition of very complex samples by comprehensive gas chromatography coupled to time-of-flight mass spectrometry. Cigarette smoke

The potential and current limitations of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC x GC-TOF-MS) for the analysis of very complex samples were studied with the separation of cigarette smoke as an example. Because of the large number of peaks in such a GC x GC chromatogram it was not possible to perform manual data processing. Instead, the GC-TOF-MS software was used to perform peak finding, deconvolution and library search in an automated fashion; this resulted in a peak table containing some 30000 peaks. Mass spectral match factors were used to evaluate the library search results. The additional use of retention indices and information from second-dimension retention times can substantially improve the identification. The combined separation power of the GC x GC-TOF-MS system and the deconvolution algorithm provide a system with a most impressive separation power.     

Determination of elemental composition of volatile organic compounds from Chinese rose oil by spectral accuracy and mass accuracy

Elemental composition determination of volatile organic compounds through high mass accuracy and isotope pattern matching could not be routinely achieved with a unit-mass resolution mass spectrometer until the recent development of the comprehensive instrument line-shape calibration technology. Through this unique technology, both m/z values and mass spectral peak shapes are calibrated simultaneously. Of fundamental importance is that calibrated mass spectra have symmetric and mathematically known peak shapes, which makes it possible to deconvolute overlapped monoisotopes and their (13)C-isotope peaks and achieve accurate mass measurements. The key experimental requirements for the measurements are to acquire true raw data in a profile or continuum mode with the acquisition threshold set to zero. A total of 13 ions from Chinese rose oil were analyzed with internal calibration. Most of the ions produced high mass accuracy of better than 5 mDa and high spectral accuracy of better than 99%. These results allow five tested ions to be identified with unique elemental compositions and the other eight ions to be determined as a top match from multiple candidates based on spectral accuracy. One of them, a coeluted component (Nerol) with m/z 154, could not be identified by conventional GC/MS (gas chromatography/mass spectrometry) and library search. Such effective determination for elemental compositions of the volatile organic compounds with a unit-mass resolution quadrupole system is obviously attributed to the significant improvement of mass accuracy. More importantly, high spectral accuracy available through the instrument line-shape calibration enables highly accurate isotope pattern recognition for unknown identification.