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Analysis of Microorganisms Using MALDI-TOF-MS A New Tool for Research Applications AXIMA-iDPlus SARAMIS Software Identification Rapid microbial identification for research use Identifies and classifies strains based on phenotype characteristics Open system allows addition of new species to the database, or the creation of new databases High performance MS and MS/MS options allow the study of microorganism proteins and peptides SuperSpectra™ reduce the incidence of false positives and ensure robustness and reproducibility Clustering allows tracking of change and evolution. AXIMA-iDPlus utilises SARAMIS: the easy to use microbial identification software which has revolutionized daily routine in analytical and diagnostic-research laboratories. The sample mass spectrum is automatically processed and condensed to a fingerprint peak list containing the mass/intensity data. This is then searched against the SuperSpectra in the SARAMIS database. AXIMA-iDPlus typically identifies 95% of clinical samples unambiguously within one minute. MALDI-TOF mass spectra of bacteria and fungi are highly taxon-specific and can be used for specimen identification. As reference spectra, AXIMA-iDPlus uses SuperSpectra which are based on mass spectra of isolates identified by widely accepted methods such as biochemical phenotyping, 16sRNA analysis or MLST (Multi-Locus Sequence Typing). Matrix Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry. The biomolecules embedded in the matrix crystals are desorbed and ionized by a laser pulse and accelerated in a high voltage field. Different ions are detected after a timeof-flight proportional to their mass, generating a high resolution mass spectrum of the sample; the ‘fingerprint’. Depending on the choice of mass spectrometer, both intact proteins (linear mode) and structural determination of fragments (reflectron mode) can be investigated. AXIMA Performance offers the same advantages as the Confidence, but with the addition of high-energy Collision Induced Dissociation (CID) to give greater fragmentation and thus better structural elucidation and sequencing for research into the proteome of microorganisms. Complementary Methods AXIMA-iDPlus is the perfect addition to the research laboratory. It meets the demands for fast and reliable screening of the broadest range of microorganisms and is complemented by biochemical methods for antibiotic susceptibility testing, by PCR diagnostic and by sequencing for unknown species using traditional MS and MS/MS methods. In addition, investigations into the lipids, glycopeptides and glycoproteins of microorganisms can be undertaken. 12 28 2 10 22 9 Clustering A006528-1: Klebsiella oxytoca AXIMA Confidence is a combination linear and reflectron mode MALDI-TOF system. In addition to microbial identification, the reflectron provides higher resolution and greater mass accuracy. The ability to perform Post Source Decay (PSD) provides the possibility to carry out MS/MS experiments, for improved structural elucidation and sequencing of peptides and proteins. 3,000 Mass (Da) Patented identification procedures are designed to definitely avoid false identifications by setting stringent thresholds for significant database matches. The analysis can be performed fully automatically or in manual acquisition mode for greater control. 94 63 88 43 7 73 111 62 56 91 62 54 AXIMA Assurance is a linear mode MALDI-TOF system with a high performance-to-price ratio. It is a robust, high sensitivity system for research use microbial identification and investigations into the proteins of microorganisms. 33 95 MALDI-TOF 41 2 47 2 30 It comprises one of the AXIMA MALDI-TOF mass spectrometers and the SARAMIS database (Spectral ARchive And Microbial Identification System). SuperSpectrum: Klebsiella oxytoca Type 9 82 56 AXIMA-iDPlus is a platform for carrying out microbial identification for research purposes and for identifying and studying the proteins of microorganisms. In uncertain cases, when the identification confidence is below 90 %, a sample’s fingerprint is re-searched against all available reference spectra in the database collected since 1998 – a significant improvement for the identification of atypical specimens. The SARAMIS software is able to carry out heirarchical clustering of samples, the results being represented by dendrograms. This technique can be used to identify changes in the microbial population over time, or to create new SuperSpectra which are representative of a particular environment. 15,000 SuperSpectrum of Klebsiella oxytoca and a fingerprint of a clinical isolate. Dotted lines indicate matching masses in the fingerprint spectrum when compared to the reference spectrum. Each SuperSpectrum is derived from multiple reference spectra of at least 15 individual isolates of a species, acquired after varying growth times and from different growth media to ensure robustness and reproducibility. SARAMIS offers a comprehensive database of species (supported by internationally recognized strain collections and clinical laboratories) and the flexibility for the user to create new entries and databases. Cultured microorganisms can be directly applied to the MALDI sample target and introduced into one of the AXIMA series mass spectrometers. Analysis and identification can be completed in 1-2 minutes. The software identifies gram-positive and gramnegative bacteria, yeasts, fungi and spores based on their characteristic MALDI-TOF-MS fingerprints down to sub-species level. No preselection of analytical methods is required; hands-on time is reduced and costs are cut dramatically. Example of a clustering of clinical samples based on MALDI-TOF mass spectra. An explicit separation is given for all species and a SuperSpectrum could be generated from all spectra of one species which could be seen as representative for the environment. Workflow Step 1: Typical Sample Preparation AXIMA-iDPlus identifies a sample grown overnight on a culture plate. A small number of cells is transferred to a FlexiMass-DS™ disposable polymeric target, followed by the addition of a matrix solution. The sample is then air-dried. 1 Minute Sample Transfer Applications AXIMA-iDPlus covers a wide range of applications. Various industries benefit from this new analytical approach, such as diagnostics research, pharmaceutical industry, healthcare and food industry. In the drug discovery process, AXIMA-iDPlus recognizes new isolates generated by microorganisms and classifies the unknown into a taxonomic tree. For use in culture collections, AXIMA-iDPlus identifies and classifies strains based on phenotype characteristics. AXIMA-iDPlus can also be used to develop manufacturing processes for the food and beverage industry; to maintain quality and to recognize upcoming toxic microorganisms at an early stage. References “Tapping the potential of intact cell mass spectrometry with a combined data analytical approach applied to Yersinia spp.: Detection, differentiation and identification of Y. pestis.” Wittwer et al. Syst Appl Microbiol. 34(1):12-9. 2011 “Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.” Bergeron et al. Eur J Clin Microbiol Infect Dis. (3):343-54. 2011 “The use in routine of mass spectrometry in a hospital microbiology laboratory.” Gravet et al. Pathol Biol (Paris). 59(1):19-25. 2011 Step 2: Identification The FlexiMass-DS target is loaded into the AXIMA where the mass spectral ‘fingerprints’ of the samples are acquired. These fingerprints are matched against the SARAMIS database of SuperSpectra for immediate identification. Results are displayed within 1-2 minutes. “Rapid genus- and species-specific identification of Cronobacter spp. by matrix-assisted laser desorption ionization-time of flight mass spectrometry.” Stephan et al. J Clin Microbiol. 48(8):2846-51. 2010. “Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI-TOF mass spectrometry.” Erhard et al. Exp Dermatol. 17(4):356-61. 2008 FOR RESEARCH USE ONLY SARAMIS is covered by European Patent numbers EP1437673 and EP1253622 SARAMIS is a trademark of bioMérieux. No attempt is made to supercede this or any other trademarks 1-2 Minute Identification Kratos Analytical Ltd. Wharfside, Trafford Wharf Road Manchester M17 1GP, UK Phone:+44 161 888 4400, Fax: +44 161 888 4402 www.SSI.shimadzu.com MO347 V1 ©Shimadzu June 2011