»Volume 2016 Issue 01 (April)
MALDI-UP – An Internet Platform for the Exchange of MALDI-TOF Mass Spectra
User guide for http://maldi-up.ua-bw.de/
J. Rau1, T. Eisenberg2, A. Männig1, C. Wind3, P. Lasch4, R. Sting1
1Chemisches und Veterinäruntersuchungsamt Stuttgart
Schaflandstraße 3/2
70736 Fellbach, Germany
Phone: +49 711 3426 1264
Fax: +49 711 588176
Email: joerg.rau@cvuas.bwl.de
2Landesbetrieb Hessisches Landeslabor (Hessian State Laboratory),
Department of Veterinary Medicine, Schubertstraße 60 Haus 13, 35392 Gießen, Germany
3Chemisches und Veterinäruntersuchungsamt Freiburg, Am Moosweiher 2, 79108 Freiburg, Germany
4Robert Koch-Institut, Proteomics and Spectroscopy (ZBS6), Seestraße 10, 13353 Berlin, Germany
Keywords:
MALDI-TOF MS, database, contact platform, spectrometry, spectra
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Abstract
Recent years have seen substantial progress towards the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in routine microbiological diagnostics. MALDI-TOF MS-based identification relies on the reproducible detection of large biopolymer mass patterns obtained from proteins of whole cells.
Besides species identification in microbiological diagnostics, MALDI-TOF MS can also be used for protein profiling, to screen for metabolites and contaminants, for epidemiological traceback, for phenotyping resistance and virulence patterns, or for biomarker detection directly from clinical material to mention only a few of the applications.
The fundamental principle of MALDI-TOF MS-based microbial identification analysis relies on a computer-based comparison of mass spectra recorded from unknown pathogens with databases containing mass spectra from samples with a known taxonomic status. Such databases are commercially available and most of the employed software and database solutions allow creating and including custom, user-specific and transferable database entries. The goal of the present work is to introduce a novel user-to-user internet platform – MALDI-User-Platform (MALDI-UP) – which was specifically designed to facilitate the exchange of information about user-specific database entries. Thereby, diagnostic gaps could be closed efficiently and databases for yet under-reported applications, such as the species identification of animal meat, insects or mushrooms via MALDI-TOF MS, could be promoted.
Introduction
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful analytical tool in food analysis, veterinary and clinical diagnostics. At the present stage the technique is used mainly for the differentiation, classification and identification of bacteria and other microorganisms in several fields of microbiology [Clark et al., 2013; Randall et al, 2015, Singhal et al., 2015, Zimmermann, 2015]. In contrast to classical microbial identification techniques, like biochemical identifications and molecular approaches, MALDI-TOF MS measurements can be carried out within minutes, coupled with the advantage of minimal material costs per identification [Dihman et al., 2011; Tran et al., 2015]. The method is not restricted to bacterial identification, but can also be utilized to determine eukaryotic cells, fungi, algae, protozoans and insects. MALDI-TOF MS was found to be useful even for species identification based on host proteins [Singhal et al., 2015; Barbano et al., 2015; Cassagne et al., 2014].
The comparison of experimental mass spectra obtained from unidentified samples (e.g. microorganisms) with reference spectra in a given database results in a ranking list containing the best matching database entries. For this approach, commercial databases use several thousands of spectra from broad microbial fields. The success of identification depends on the quality and the species coverage of the entries included in the used database. Manufacturers place large efforts on extending their databases. However, due to economic reasons, the time between validated manufacturer updates can be long. The systems (consisting of hardware, software and the MS database) employed by most users do not only offer the gradual update of an extensive database supplied by the manufacturer, but also allow for the addition of custom data. Such data can be transferred within the device platform, making it possible to close diagnostic gaps more quickly. In medical and veterinary microbiology, this is particularly relevant for various special microorganisms and highly pathogenic species. Although there is a great demand, until now, there are only a few MALDI-TOF MS database entries available for non-clinical applications, such as the species identification of animal meat or of fish.
The reliable exchange of custom database entries requires that the user who creates the entry (creator) provides certain basic information (metadata) to the user who receives the data (recipient). The recipient is primarily interested in what is being offered and of what quality the entry is: Briefly, this aspect covers the accuracy of species assignment by reference methods, the quality of sample preparation, spectra acquisition, selection, and processing.
Only if this basic information (metadata) is reliable, the recipients will accept such an entry and add it to existing MS databases. Therefore, the recipients must be able to identify the creator of the entry. This can be achieved either through personal contacts, or by means of an open information exchange platform. The online MALDI-UP catalog which is available at http://maldi-up.ua-bw.de and which is hosted by the Chemisches und Veterinäruntersuchungsamt Stuttgart (CVUAS) was specifically designed for this purpose. The catalog lists user-created database entries that are uniformly presented and contains comprehensive metadata information such as detailed taxonomic information, isolate numbers and sample preparation parameters. Furthermore, the designations of isolates (e.g. molecular identification, biochemical data) as well as technical details of the entries (instrument, cultivation, preparation, etc.) are given. The summary of database entries and the contact information of the creators of the entries are also presented. The MALDI-UP catalog is not intended to provide free downloads of the spectra. This is important because it assures that the protection of copyrights will remain in the hands of the copyrights owners.
Usage of the catalog and description of the procedure to add spectral entries
The MALDI-UP catalog is open to everyone interested in providing information about personal database entries with the option to exchange or distribute spectra under own merchandising criteria. The catalog can be used, subject to the conditions stated on the homepage. It is also a source of information for everyone who wishes to contact spectra creators.
The catalog (in Microsoft Excel) can be downloaded from the MALDI-UP platform and new data entries can be added in the proposed format. Following the submission of personal new data to maldi-up@ua-bw.de, they will be carefully checked and missing information will be requested. Finally, the latest entries will be included in the next update of the catalog. Creators agree to make their contact information publicly available (institutional and email address) and principally agree to respond to data requests. The procedure ensures that the creators themselves control the exchange of information with their colleagues.
The catalog will be continuously updated on a regular basis, at least monthly, and demands a procedure of disclosed parameters for validation. This concept was developed with assistance from employees at CVUA Stuttgart and the Hessian State Laboratory (for comments and suggestions please contact maldi-up@ua-bw.de).
The MALDI-UP catalog – a detailed description
The following section specifies the information given in the spreadsheets of the MALDI-UP catalog, including relevant column headings.
Spreadsheet Entries – Information fields (Fig. 1)
In the upper part of the spreadsheet one can find contact information to the editor of the version (defined by the number of entries and the date). There is also a link to the MALDI-UP homepage to get back to the main website. The next rows include the column titles of the table in English and German. The row “FILTER” serves as an Excel filter for each column to facilitate the individual search and adaption of the table after download.
The entries are sorted by a rough taxonomic classification. Following the download of the MALDI-UP catalog, the sorting can be restricted and controlled using the filters.
The columns of the table were grouped in six sections:
| Basic data for the entry | |||||
| Taxonomic information about the used material | |||||
| Data for the isolate or material | |||||
| Technical data for the entry | |||||
| Results of an independent sample preparation in routine mode, using the new entry for identification | |||||
| Information about the number of users who have tested the entry and the type of feedback (positive/ negative) | |||||
Explanation for every data column:
| Basic data for the entry | |
| No. | Serial number of the database entry |
| Date | Listing date |
| System | Mass spectrometry system used |
| User responsible for the line (see contact) | Institution responsible for the information in this row. The contact information of the creator is given on the spreadsheet "contact". |
| Taxonomic information about the used material | |
| Domain | Rough classification in taxonomic criteria up to subspecies-level to facilitate searching and filtering |
| Phylum | |
| Class | |
| Order | |
| Family | |
| Genus | |
| Species | |
| Subspecies | |
| Data for the isolate/material | |
| Identifier | Identifier of the isolate, preferably a number of a public strain collection (e.g. DSM, ATCC …) or an isolate collection number (e.g. of the creators CVUAS 1234) |
| Isolation source / date / [reference] | Source of the material, literature reference (if available) |
| Isolate tracking | Known history of the isolate. Isolates should be conserved to allow subsequent verification. |
| Further details on the isolate | |
| Spore former | The ability of the isolate to form spores, because of the possible influence on MALDI-TOF MS spectra and preparation requirements. |
| Verification of assignment | Principle of the verification of the isolate / material (e.g. sequenced, type strain, veterinary post mortem pathology examination |
| Results of sequencing | In this field a short version of sequencing results can be provided, which supports the species assignation. Depending on the primer-system used, sequencing results are not always the sequence of the whole target gene (e.g. 16S rDNA, rpoB, gyrB). The resulting match with the first reliable hit in a genetic sequence database should therefore be given with the number of bases. If available in format: 16S rDNA: 1387bp (96.70% of ref-sequence) > 1[difference bp]/1387 = 99.93% similarity with Flavobacterium psychrophilum JIP02/86, 100% completeness (11.06.15; EzBioCloud) |
| Other results for assignment of the isolate | E.g. biochemical data, or specific PCR results, especially important for species decision (if available) |
| Isolate / material conserved as | Own isolate collection or reference to public strain collection. |
| Technical data for the entry | |
| Agar / medium | Agar (e.g. SBA = sheep blood agar) used for cultivation of the microorganism |
| Kind of medium | Solid / liquid |
| Cultivation temperature | In degree Celsius |
| Cultivation time | In hours (h) or days (d) |
| Cultiv ation, other conditions | e.g. atmospheric conditions (aerobic, anaerobic) |
| Sample preparation [reference] | Sample preparation protocol used, with reference if available |
| Matrix | e.g. HCCA |
| Creation of the entry according to the manufacturer's specifications by trained personnel | Yes or no |
| Full name of entry | Full name of the new entry |
| Date of entry (year/day/month) | Creation date of the new entry |
| Results with the entry | |
| Result of an independent sample preparation in routine use | |
| Sample preparation > date of independent spectrum | e.g. direct transfer (DT); date in format day/month/year |
| Commercial database version | Commercial database version used (e.g. DB 5898) |
| Result with commercial database version | Result of the independent spectrum identified with the commercial database e.g. Biotyper-format: Staphylococcus aureus (1.782) B: 1.782 is the score value, A/B/C the consistency category of the Genus / Species according to [Anonymous, 2012] |
| Result with the new entry | Result of the independent spectrum identified with the database including the new entry |
| Remarks for interpretation | Remarks or information about the interpretation of this entry (e.g. “closely related to Staphylococcus aureus”) |
| Information about the number of users who have tested the entry and the type of feedback (positive/ negative) | |
| Feedback from other users of the entry | |
| Number of known external users of the entry | Number of recipients, with whom the entry has been shared |
| Number of users who gave feedback about the entry. Green: positive experiences; red: negative experiences | Number of reported experiences for the entry: positive experiences confirm the result found with the new entry; negative experiences fail to confirm the new entry |
Spreadsheet Contact (Fig. 2)
The spreadsheet “contact” contains addresses of creators and their institutions, in alphabetical order, who have deposited information on own database entries.
The Row “FILTER” serves as Excel-filter for every column, to facilitate the individual search and adaption of the table after download.
| Institution | Name in abbreviated form |
| Name in short form | |
| Logo of the institution | |
| Address | Official name of institution |
| Street | |
| Area code and city | |
| Country code | |
| E-mail institution | E-mail address of the institute, without one specific recipient |
| Contact person | Physical person, who works with the MALDI-TOF MS / provides entries |
| E-mail contact person | Email address of the contact person |
| MALDI-System | The type of MALDI-TOF MS equipment available |
| Institution category | Private / Governmental / Inspection service… |
Spreadsheet References (Fig. 3)
This page summarizes the references used for the identification of certain species. It also refers to the extraction methods used. Sequential numbers are given, followed by a column with the complete citation of the article.
If a new reference is essential for the creator, it should be submitted together with the database entry. The reference number will be edited by the webmaster of the MALDI-UP list.
Speadsheet History (Fig. 4)
This page provides an overview of the history of uploads and the total sum of entries. Additionally, it states the next scheduled update of the list.
Row 3 contains several columns:
| Date | Chronological list of update-dates |
| Number of institutes | Number of institutes that have provided information about database entries (see page contact) |
| Entries sum | Total number of entries |
| Microbes | Number of entries for microorganisms |
| Meat | Number of entries for meat |
| Fungi | Number of entries for fungi |
| Still open, for other types of entries (e.g. plants, algae etc.) |
This sheet also contains a graphic overview of the progression of entries [Fig. 4].
Discussion
Universal use of databases depends on size and quality of data entries. The purpose of the MALDI-User-Platform (MALDI-UP) is to provide MALDI-TOF MS users with an easily accessible possibility to exchange database entries. At the time of writing (March 2016), altogether 133 entries by users of seven institutions have been added to the catalog (Version 133/18.03.2016) since its first release (September 2015). These database entries originate from different mass spectrometers (e.g. Bruker LT microflex and autoflex, and bioMeriéux VITEK MS Plus). Furthermore, since the database has been launched in November 2015 the number of downloads has increased continuously (Fig. 4).
This open access catalog does not claim to substitute the manufacturers’ continued efforts to provide database extensions. It allows, however, to gain a quick overview on available database entries from users working in the same area of research or diagnostics with special interests, such as animal meat, insects or mushrooms. Furthermore, the contact to colleagues can be easily established, independently of whether the creator is from a research institution, a hospital or an official or private diagnostic laboratory. The interest during the first months is reflected by the number of deposited entries and the growing number of downloads in the last months (Fig. 4, Fig. 5).
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The interest in the catalog and its benefit for the user will increase for both, creators and recipients, as the number of approved data entries increases. Additionally, fruitful collaborations can arise beyond the exchange of database entries alone.
The potential of finding interested creators, capable of offering high-quality entries for exchange, is considered high, for the following reasons:
- the number of marketed MALDI-TOF mass spectrometers increases world-wide (2015 > 2.000),
- a higher level of training of MALDI-users, offered by the manufacturers or other institutions, ensures an increase in knowledge and
- a wide range of research projects deal with MALDI-TOF MS use and validation.
Increasing numbers of catalog entries and database downloads are promising and an incentive to consolidate the MALDI-UP project as a source for current data as a pillar of up-to-date diagnostics. We have already proven that MALDI-TOF MS can be a suitable tool to be integrated in the description of novel species. Therefore, it is increasingly important to quickly provide MS data of newly detected species. This has been common practice for several years for data on taxonomically important genetic sequences. As an example, we refer to the MALDI-UP catalog entries for the recently described bacterial species Bacillus cytotoxicus CVUAS 2833 (entry 0001; Guinebretière et al., 2013) and Listeria fleischmanni (entry 0006; Bertsch et al., 2013) as well as for the entries of Streptobacillus (S.) felis (entry 0013, Eisenberg et al., 2015 a), S. notomytis (entry 0119; Eisenberg et al., 2016 a) and S. ratti (entry 0120; Eisenberg et al., 2016 b).
Additionally, the database focused on the identification of species of food animals has recently been augmented by spectral data for pork meat (entry 0004; Stoll et al, 2015).
In conclusion, the launch of the MALDI-UP catalog aims to support networking and expansion of the community of MALDI users based on mutual sharing of information.
Acknowledgements
The authors like to thank M. Contzen, E. Hiller (CVUAS), and Andy Schneider (RKI Berlin) f or helpful discussions and S. Böttcher (CVUAS) for preparing the MALDI-UP web-page.
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End of Article.
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