Goslar, 09/10/2007 Identification of Microorganisms using MALDI-TOF MS profiling: Adopted sample preparation methods and bioinformatic approaches Dr. Markus Kostrzewa Bruker Daltonik GmbH, Leipzig
Goslar, 09/10/2007 MALDI-TOF MS microorganism identification Identified species Select a colony Prepare onto a MALDI target plate Unknown microrganism ? Data interpretation Generate MALDI-TOF profile spectrum
Goslar, 09/10/2007 Target/Acceleration Time-of-FlightMolecular MassDesorption/Ionisation DetectorDrift Region m/z a.i. Mass Spectra Laser MALDI-TOF mass spectrometry
Goslar, 09/10/2007 Sample preparation Direct “cell smear“ method most simple method, applicable to many bacteria Organic solvent extraction improved quality for difficult bacteria, yeast, fungi Mechanical cell disruption (e.g. sonication) In case of very ridgid cell walls Compatibility of different procedures Protocols for inactivation and shipment of microorganisms are available
Goslar, 09/10/2007 E.coli MALDI-TOF MS profile spectrum Positive linear mode Mass range 2-20 kDa
Goslar, 09/10/2007 Arthrobacter, effect of storage products Taken from: Vargha M et al. J Microbiol Methods. 2006 Possible influence of growth state 16h 24h 64h 0 1000 2000 3000 4000 3000400050006000700080009000100001100012000 m/z Clostridium butyricum, effect of sporulation Coop. with Prof. Krüger, Dr. Grosse-Herrenthey, Leipzig, Germany
Goslar, 09/10/2007 MALDI BioTyper 2.0 - GUI Unknown samples Match against microbial reference database Identification result
Goslar, 09/10/2007 MALDI BioTyper 2.0 – realtime analysis Wizard guiding through setup from measurement to data analysis BioTyper Automation Control Wizard Define Project Analyte Placement Select Methods Start MALDI BioTyper 2.0: realtime Analysis Start Automation Control Wizard. Use a SOP and do not bother with instrument settings. Results available directly after measurement
Goslar, 09/10/2007 Score based pattern matching Calculation of a matching score based on: Rel Score % matches of the reference spectrum (e.g. 6 / 10 = 0.6) Rel P-Num. % matches of the unknown spectrum (e.g. 6 / 20 = 0.3) I-Corr. value of intensity correlation Unknown microorganism is matched against each Main spectrum in a library. Ranking according to matching score, threshold for ID Robust standard method for species ID
Goslar, 09/10/2007 Neisseria meningitidis serotypes A W135 X Y How about subtyping?
Goslar, 09/10/2007 Incorrect hirachical clustering of three Neisseria meningitidis serogroups after PCA Principle component analysis PCA is looking for the largest variations in a given group. If measurement variations are larger than inter-species/ subspecies variations it may fail! Depending strongly on standardization of measurement!
Goslar, 09/10/2007 Weighted pattern matching Batch weighting: Automated generation of a weighted main spectra library; every main spectrum of a library is compared with all the other main spectra Manual weighting: Weight of each peak in a main spectrum can be edited manually Combination of both procedures is possible Hierachical approach in combination with standard pattern matching Characteristic peaks are selected and weighted by occurence in subgroups, intensity, and frequency
Goslar, 09/10/2007 Minimal sample preparation Powerful bioinformatic approaches Species to strain resolution, mixture detection High throughput at low costs per analysis Non-expert identification possible Dedicated databases of high quality Conclusions
Goslar, 09/10/2007 The BDAL BioTyper team: Thomas Maier Kristina Schlosser Thomas Wenzel Thorsten Mieruch Stefan Klepel Uwe Renner Jan-Henner Wurmbach Karl-Otto Kräuter Alexander Rueegg Thanks to: … and many cooperation partners! In particular: Prof. Stackebrandt, Dr. Schumann