Phylogenetic classification and identification of bacteria by mass spectrometry

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1 Phylogenetic classification and identification of bacteria by mass spectrometry Anja Freiwald & Sascha Sauer Max Planck Institute for Molecular Genetics, Otto-Warburg-Laboratory, Berlin, Germany. Correspondence should be addressed to S.S. Published online 23 April 2009; doi: /nprot Bacteria are a convenient source of intrinsic marker proteins, which can be detected efficiently by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. The patterns of protein masses observed can be used for accurate classification and identification of bacteria. Key to the reliability of the method is a robust and standardized procedure for sample preparations, including bacterial culturing, chemical treatment for bacterial cell wall disruption and for protein extraction, and mass spectrometry analysis. The protocol is an excellent alternative to classical microbiological classification and identification procedures, requiring minimal sample preparation efforts and costs. Without cell culturing, the protocol takes in general o1 h. INTRODUCTION Efficient methods for classification and identification of bacteria are important for many applications in microbiology 1. Depending on the application and the bacteria under investigation, in many cases, laborious traditional methods comprising serological, physiological, biochemical and chemotaxonomic, or more modern genomic procedures are being used 1. Particularly, traditional methods such as ELISA are often only designed for specific bacterial species or the Analytical Profile Index is only applicable to a limited number of bacteria 1. Mass spectrometry has become an important tool in the life sciences and in diagnostics 2,3. Owing to the specificity, speed of analysis and low costs for consumables, a standardized procedure for mass pattern analysis by matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry for classifying and identifying bacterial species as is presented here can replace a number of conventional methods 4 8. The mass spectrometry protocol offers an attractive alternative to traditional laboratory protocols for classification and identification of microorganisms in a variety of areas, such as medical diagnostics, food and water control, and environmental research. In contrast to DNA-based methods, the detection of mass patterns by mass spectrometry does not require reference genome or protein sequences. Patterns of protein masses (i.e., the mass signals and the mass signal intensities) detected by MALDI mass spectrometry can be analyzed efficiently in high throughput using, e.g., hierarchical clustering, pattern-matching or more sophisticated algorithms such as support vector machines 9,10. As compared with protein identification-based mass spectrometry procedures that are widely applied in the basic life sciences and require high mass accuracy for reliable identification of proteins 3, pattern analysis methods are more robust against slight mass deviations detected using the MALDI mass spectrometer as is shown in this protocol (Fig. 1). A large majority of the bacterial proteins and fragments detected by the approach shown here are of ribosomal origin (Fig. 2) (ref. 11). This can be attributed to the high abundance of ribosomal proteins in the bacteria and to the protocol used, which favors the extraction and detection of these basic proteins or fragments thereof. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations occur even at the subspecies and strain level. Ribosomal proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates. The different masses of these ribosomal proteins represent a specific fingerprint that is useful for applying unsupervised hierarchical clustering approaches or using model construction methods 12. These approaches can be used for phylogenetic classification of bacteria such as genomic 16S rrna sequences 1. In practice, however, complete bacterial genomic or protein sequences are often not available, which inhibits the application of PCR-based approaches. In general, DNA-based methods such as PCR require optimization for setting up specific assays for each bacterial strain. Once they are being set up, specific genomic procedures, such as real-time PCR, are faster than the procedure described in this protocol, as they do not necessarily require cell culturing. However, for example, the vast majority of the illnesses and 64% of the deaths caused by bacterial contamination of food are from unknown and badly characterized microorganisms 13. Mass spectra of these bacteria could easily be produced, and spectra can be stored for the identification of disease-causing bacteria. Thus, in many practical cases in microbiology, rapid and specific pattern-matching procedures can be very useful for the classification and identification of harmful bacteria, where no sequences are available. Although DNA sequencing is the current gold standard for molecular characterization of bacteria, this genomic method cannot be easily applied for fast classification and identification. Bacterial sample Ethanol formic acid extraction 80% TFA extraction Direct transfer Mass spectrometry Computational analysis Figure 1 A general overview of the procedure. A bacterial sample is subjected to a protein extraction method and then analyzed by mass spectrometry. Pattern-matching analysis software of mass spectra allows identifying bacterial species and subspecies unambiguously. 732 VOL.4 NO NATURE PROTOCOLS

2 The standardized bacterial detection procedure presented herein is facile and reproducible. It fulfils biological safety requirements and can be easily scaled up. Owing to the software and algorithms described in Box 1, robust high-throughput mass spectrometry analysis of bacteria can be performed using simple preparation protocols. The methodology has proved sufficient maturity for easy and systematic application in many fields of microbiology. Due to its analytical robustness and simplicity, the protocol described here can be easily adapted by a microbiologist with basic knowledge in mass spectrometry. The main application of the method applies to bacteria, although the procedure can also be extended for the analysis of yeast and fungi. A library of B3,000 bacterial reference mass spectra has been established with the approach described in this protocol 14 and it is being applied in a growing number of microbiology laboratories 15,16. In this article, we show a detailed, step-by-step protocol for facile mass spectrometry analysis of bacteria (Fig. 1). The method can be adapted to the requirements of the respective microbiology laboratories. The protocol includes the following: (i) general information on bacterial culturing conditions and an exemplary procedure as shown for culturing Enterobacteriaceae, (ii) three procedures for chemical treatment for extracting proteins from whole bacterial cells and (iii) subsequent automated MALDI-TOF mass spectrometry detection. Moreover, we describe how (iv) to generate reference mass spectra and (v) to classify and identify bacterial samples. Several parameters can affect the reproducibility of protein mass pattern detected in bacteria, which includes sample preparation, growth phase, culture conditions, sample storage and slightly varying performances between different MALDI-TOF instruments (see also Experimental design). Therefore, the user of this protocol should consider that all these experimental differences can influence the reproducibility and reliability of the method. Although these experimental differences in general do not significantly hamper identification on the bacterial species level, the distinction on the subspecies level might become problematic when results of different experimental setups are being compared with each other. Depending on the procedure of chemical treatment applied for the extraction of proteins, bacterial cells are required for efficient MALDI-TOF detection. This number of cells can be easily produced by cell culturing. Culturing bacteria on solid media also reduces the sample complexity and improves the control of environmental bacterial samples. Identification of bacteria by matching protein mass patterns is largely independent from culturing conditions, particularly on the species level, but clearly, this depends on the (sub-) species analyzed 14. However, potential culture dependency of some mass signals in the spectra can be excluded by using sophisticated data analysis software 14.Inany case, we recommend comparing mass spectra of clones of bacteria that were grown under similar conditions, including media and growth times. For the chemical treatment of bacterial colonies to allow the extraction of proteins for the MALDI-TOF analysis, we present the following three approaches: Intens. [a.u.] RL ,000 3,000 4,000 RS22 RL34 RL33meth RL32 RL35 RL29 RL30 RL31 RS21 RS19 RS15 5,000 6,000 7,000 8,000 9,000 10,000 11,000 m/z Figure 2 A mass spectrum of an E. coli sample. Many of the mass peaks are derived from proteins of ribosomal origin 20.SpectraofE. coli also serve as a mass calibration control in the protocol. (i) The ethanol formic acid procedure is our standard and most frequently applied approach that works for a large variety of bacteria 14. This protocol is suitable for inactivating potentially pathogenic bacteria without spore formation. Ethanol is used for inactivation of the bacterial samples. Treating inactivated bacteria with inexpensive chemicals such as formic acid for cell wall disruption and acetonitrile for protein extraction involves short incubation and centrifugation steps. Approximately bacterial cells are required for this approach. (ii) The trifluoroacetic acid (TFA) procedure is an alternative to the ethanol formic acid method for bacteria with spore formation (such as bacilli) or for highly pathogenic bacteria that do not completely die off after ethanol treatment. Using TFA treatment, inactivation and cell wall disruption are performed in one step 17. (iii) The direct transfer method is by far the most convenient and fastest approach: a single colony is smeared on a MALDI target position as a thin film. Inactivation of bacteria takes place while the samples are prepared on the MALDI target plate with organic components of the MALDI matrix solution such as acetonitrile and TFA. However, the user has to consider whether this procedure completely fulfils biosafety regulations for the particular application. Moreover, with some (soil) bacteria such as Actinomycetes and Norcadia, better results can be produced using the ethanol formic acid extraction method. The limitation of the protocol consists in the need of cell culturing to produce sufficient amounts of bacterial cells and to reduce the sample complexity. The strength of the protocol is its striking simplicity that allows easy application in microbiology laboratories equipped with a MALDI mass spectrometer. The experimental steps described in the protocol can be easily scaled up in a well-plate format. To detect protein mass patterns, the protocol requires only a simple linear ion mode MALDI-TOF mass spectrometer that allows automatic measurements. For phylogenetic analysis and reliable identification of bacteria, the method benefits from the use of advanced bioinformatics analysis as is described in Box 1 (ref. 14). NATURE PROTOCOLS VOL.4 NO

3 BOX 1 COMPUTATIONAL TOOLS FOR BACTERIAL PROTEIN MASS PATTERN ANALYSIS For bacterial data analysis, we use the MALDI Biotyper software (Bruker Daltonics). Using this software, we pre-process spectra by applying default parameters for reference spectra libraries, which are termed main spectra libraries (MSPs). Thereby, we apply mass spectra compressing, the Savitsky Golay smoothing method and multipolygon correction method (two runs). For data normalization, we use maximum norm and spectra differentiation to search mass signals with a signal-to-noise (S/N) ratio of 3 and select maximally 100 mass peaks with a threshold of in the mass range of 3,000 15,000 Da. The main spectra are subsequently generated and saved as a reference for a specific bacterial species using all spectra given for a single bacterium. Therefore, 75 mass signals that occur in at least 25% of the spectra with a mass deviation of 200 p.p.m. are selected automatically. Different libraries of bacterial reference mass spectra can be opened using a set-editor of the MALDI Biotyper software. The user can load libraries of choice and either add another library to combine them or open a second library and copy only the spectra of selected microorganisms. For phylogenetic analysis, we cluster hierarchically mass spectra of bacterial species and subspecies. Each mass spectrum of a dataset is compared with the other spectra. This procedure results in a matrix of cross-wise identification values. The matrix is used to calculate the distance values for each pair. On the basis of the distance values obtained, we produced a dendrogram using the appropriate function of the statistical toolbox of Matlab 7.1 (The MathWorks Inc.). Taking a list of mass signals and their intensities into consideration, dendrograms are produced by a similar scoring of a set of mass spectra. Dendrograms show graphical distance values between species constructed from their reference mass spectra. A correlation function is applied for calculating distance values. For graphical correlations, an average statistical algorithm is implemented in the MALDI Biotyper software. The arrangement of bacteria on the left side of the dendrogram (Fig. 3) is arbitrary. In general, species with distance levels below a value of 500 are reliably classified. This value can be changed by the user, however. For initial species identification, we apply pattern-matching algorithms. Reference spectra of a compiled library and mass spectra of bacterial samples for identification are loaded. The spectra are marked and the software starts identification by pressing the following commands: Identification, MSP Projection Scores and Run. Then, the pattern-matching algorithm calculates calibrated m/z values, average intensities and frequency distributions of each mass signal in different measurements. The results appear for all mass spectra marked with a score value. For identification score, the user switches to result list. For identity scoring, the algorithm implemented in the analysis software counts signals in experimental mass spectra that match with reference mass spectra and vice versa. Before matching experimental mass spectra with spectra from the reference libraries, the software calibrates using the quadratic calibration function spectra with each other, i.e., the software tries to produce as minimal mass deviations as possible. If two mass spectra in the mass range of 3,000 15,000 Da show, e.g., mass deviations of signals of about 1 2 Da, the signals will match much more precisely after the calibration procedure. We found that reliable pattern matching can be performed using this quadratic calibration procedure and also a mass tolerance of 200 p.p.m. in the mass range of about 3,000 15,000 Da (ref. 14). For identification scoring, the software correlates signal intensities of matched signals of mass spectra. The three scores obtained from such a procedure are multiplied and normalized to a value of 1,000 and its common logarithm. Log scores over 2 are considered as reliable identification of a bacterial species (Table 4), whereas log scores over 1.7 generally indicate reliable identification of bacterial genera. Log scores of 3 are obtained when spectra are matched with themselves. For reliable identification of closely related bacterial subspecies, we use an advanced weighted pattern-matching algorithm. In this refined approach, we assign additional values to informative mass signals that we observe in the reference spectra of bacterial subspecies 14. Clearly, the mass spectra analysis for bacterial samples is not restricted to the use of the described software package that is applied in our laboratory. Instead of hierarchical clustering, it was recently shown that model construction can also be applied, including quick classifier (QC), support vector machine (SVM) and genetic algorithm (GA) 12. Alternatively, classification algorithms, such as the supervised neural gas and the fuzzy-labeled self-organizing map, might become useful 21. The user might also test alternative classification procedures and software from companies such as MicrobeLynx 22 from Waters ( the BioNumerics software (version 3.5; Applied Maths, Kortrijk) 23, statistical tools such as Matlab or methods that are described in ref. 24. Moreover, specific software for bacterial identification and phylogenetic analysis was developed by a number of academic research groups For more information on the alternative software available, the reader is referred to refs and the following website: In general, mass spectrometric data from many different MALDI instruments can be exported and loaded into software packages, e.g., in XML or ASCII format. Experimental design Wet-lab procedure. The first part of the protocol deals with culturing conditions. The efficiency of bacterial cell culturing depends significantly on the bacteria under investigation. To show a complete workflow, we have chosen culturing conditions that work well for Enterobacteriaceae, which we primarily analyze in our laboratory. For Enterobacteriaceae,inmostcases,Luria-Bertaniwith 1% (vol/vol) glucose is a suitable medium for culturing. For many other bacteria, the user should apply appropriate culturing procedures and proceed with chemical treatment or the direct transfer procedure. A large variety of conventional liquid media and growth conditions can be applied depending on the species under investigation. For example, in clinics, blood media are commonly used to culture bacterial species such as Neisseria gonorrhoeae. Bacterial colonies grown on solid media might be more heterogeneous than those grown in liquid culture. However, in general, the mass spectra of bacterial subspecies either grown on solid media or in liquid culture can be easily compared with each other. In any case, the user should avoid analyzing (heterogeneous) bacterial samples that are still in the lag phase or bacteria that entered the death phase. The user should also avoid contamination of different clones growing on the agar plate, as this can affect the mass spectra analysis later on. The presence of culture medium adhering to the bacterial colony cells from solid medium such as agar has no visible effect on the mass signal patterns. However, to avoid any potential problems in the analysis later on, contamination of medium should be minimized by accurately picking the bacterial clones. 734 VOL.4 NO NATURE PROTOCOLS

4 BOX 2 MALDI-TOF MEASUREMENT (AN EXAMPLE WITH THE ULTRAFLEX MASS SPECTROMETER) PROTOCOL 1. First, create an Excel data file containing general parameters such as target geometry, automatic measurement method and information on position, directory and folder name for mass spectral data saving. 2. Convert Excel file into.txt by saving as Text (Tab delimited)(*.txt). 3. Copy to the AutoXecute folder and load into FlexControl Software. Check if folder names for saving are permitted. 4. In general, we use the following parameters: linear positive ion mode, mass range from 2,000 to 20,000 m/z with suppression up to 1,500 m/z. Ion source 1 is set to kev, ion source 2: kev and lens to 6.00 kev. Pulsed ion extraction (PIE) is set to 0 (ns). Save all FlexControl parameters in a FlexControlMethod. 5. Load a target plate into the mass spectrometer. 6. Go to the first spot on the MALDI plate. 7. Determine lowest laser power intensity, which gives a reasonable number of mass signals. 8. Change the AutoXecute method settings according to your experience in Step 7 and load FlexControlMethod saved in Step 4 into the AutoXecute method. 9. Direct the target plate geometrically with the MALDI-TOF instrument so that laser shots are centered to the spots of the target. 10. Start measuring. Accumulate mass spectra for 100 laser shoots per step (laser frequency is tuned to 66.7 Hz). Sum up to 1,000 mass spectra according to the following restrictions: for evaluation, use Centroid peak detection at 80% height. Mass signals in a range of 7,000 10,000 m/z that have been baseline subtracted (Convex Hull) must have a resolution higher than 600 with an signal-to-noise of 2 and an absolute signal intensity of at least 1,000. Maximal resolution is 10 times above the threshold. 11. Turn Fuzzy control on. Initial laser power should be set to percentage when signal emerges out of the background (e.g., 35%; determined in Step 7). Set maximum laser power 2 5% higher and use the initial laser power on a new raster spot. 12. Shoot a maximum of 300 times with the laser at one spot but ignore if signals are still good. Quit spot measurement after 30 fails. For the generation of reference mass spectra (main spectra libraries), the user should measure four times on one spot using four different raster layers. For identification, we generally acquire mass spectra by accumulating 1,000 laser shots in shot portions. For good quality mass spectra, only one colony should be used for the analysis. It is very important that the user always compares mass spectra of bacterial samples that were grown under similar conditions, particularly for bacterial analysis on the subspecies level. It is generally important that the bacteria analyzed for comparative mass spectrometric analysis have entered the same growth phase (use samples that are either in the log or in the stationary phase). Although chemical extraction of proteins from bacterial cultures can be performed using either of the two approaches mentioned and described in detail in Step 2. For the direct transfer procedure, bacterial samples are applied directly onto a MALDI target without any prior chemical treatment (as is described in Step 6). Mass spectrometry analysis. In principle, the protocol described here can be applied for any MALDI instrument of the Ultraflex, Autoflex or Microflex series of Bruker Daltonics and for instruments from other companies. Measurements are performed in the linear positive-ion mode, which means that no reflector device is required. In general, the linear ion mode allows for higher sensitivity than does the reflector mode, and still produces mass spectra with sufficient mass accuracy and resolution for later patternmatching analysis as described in Box 1. It is important for the efficiency of the high-throughput method that the mass spectrometer performs measurements in an automatic mode, which is the case for most instruments that have been launched on the market during the last decade. Different instruments with slightly different settings can result in slightly different mass spectra. For example, different lasers such as a nitrogen, a neodynium-doped yttrium aluminium garnet (ND:YAG) or a spatially structured ND:YAG laser in the MALDI mass spectrometer can result in different detection efficiencies 18.In Box 2, we provide an example of automatic measurement using the Ultraflex mass spectrometer equipped with a ND:YAG laser. The user might consult the example from Box 2 to adapt the protocol to his/her specific MALDI instrument. In case the user wishes to apply different MALDI-TOF instruments, we recommend using Escherichia coli standards (Box 3) and comparing the mass spectra of these samples as an initial test. The user, however, should ensure if mass spectral data from different MALDI instruments can be compiled in a single analysis. MATERIALS REAGENTS. Tryptone/peptone from casein (Carl Roth, cat. no ). Yeast extract (Carl Roth, cat. no ). Sodium chloride (Merck, cat. no ). Agar (Carl Roth, ). D-(+)-Glucose anhydrous (Carl Roth, cat. no. X997.1). Ethanol, absolute (Merck, cat. no ). Protein Calibration Standard I (Bruker Daltonics, cat. no ). a-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics, cat. no )! CAUTION Harmful if inhaled, if in contact with skin or if swallowed.. E. coli strain for calibration; we used E. coli 1100 (E. coli/k-12, Lab collection JKI Dossenheim)! CAUTION Whatever culture conditions are applied for bacteria under investigation, handle all biological samples as a potential source of pathogens. Use appropriate protective attire (lab coats, safety glasses, latex gloves and other conventional measures applied in the microbiology laboratory) and dispose properly all biohazardous materials.. Acetonitrile (Carl Roth, cat. no )! CAUTION Highly flammable and toxic. Use appropriate protective attire (lab coats, safety glasses and latex gloves) and pipette under fume hood. NATURE PROTOCOLS VOL.4 NO

5 BOX 3 REPRODUCIBILITY OF THE PROCEDURE 1. Grow five samples of E. coli as described in Step 1. Alternatively, the user can apply other bacteria available in the laboratory and grow the samples with the user s optimized protocol but according to the guidelines mentioned in the main text. 2. Prepare these five samples by applying the same chemical treatment procedure (e.g., ethanol formic acid extraction). 3. Spot five times 1 ml of each bacterial sample on different positions of the MALDI target. 4. Insert the target in the MALDI instrument. 5. Perform automated sample measurement (as described in Box 2). Measure four times on each of the five spots per bacterial sample with different laser raster layers and accumulate the respective spectra separately from each other. 6. Open spectra with the respective instrument software for examination. 7. For evaluation of mass spectra reproducibility, load the spectra into the ClinProTools 2.1/2.2 software. Through this process, mass spectra are firstly normalized before baseline subtraction, peak detection, realignment and peak area calculation are applied. The optimal settings are (i) a signal-to-noise ratio of 5, (ii) a Top Hat baseline subtraction with 10% as minimal baseline width, and (iii) a 3-cycle Savitsky Golay smoothing with a 10-Da peak width filter. 8. Calculate the coefficient of variation (CV) of intra-runs. Load the data of each single bacterial sample into ClinProTools software and run Report 4 Peak Statistic. Approximately 100 mass signals of the 20 mass spectra derived from one bacterial sample will be detected in each mass spectrum. ClinProTools automatically calculates the mean intensity value per mass signal and the respective standard deviation. The CV of each mass signal is calculated by dividing the standard deviation to the mean intensity value. The sum of the CVs of the 100 mass signals is divided by 100 to obtain an average CV value. In addition, the respective standard deviation is calculated. The intra-run CV is calculated by dividing the standard deviation of the average CV by the average CV value. 9. To calculate the CV of inter-runs, copy all 100 mass spectra of the five bacterial samples into one folder and load it into ClinProTools software. Process according to the procedure outlined in Step 8. Note: Recently, an alternative approach for the analysis of the effects of the experimental factors associated with MALDI-based detection of protein mass patterns was presented, which included analysis of variance (ANOVA) and principal component analysis (PCA) 29.. Formic acid (Sigma-Aldrich, cat. no )! CAUTION Highly corrosive. Use appropriate protective attire (lab coats, safety glasses and latex gloves) and pipette under fume hood.. TFA (Fluka, cat. no )! CAUTION Highly corrosive. Use appropriate protective attire (lab coats, safety glasses and latex gloves) and pipette under fume hood.. Liquid soap (e.g., GH11 dish liquid 1 liter (Grasshopper, cat. no )). Water LC-MS CHROMASOLV (Fluka, cat. no ) EQUIPMENT. Petri dish (Greiner, cat. no ). Propylene tube 14 ml (Greiner, cat. no ). Inoculation loop 1 ml (Carl Roth, cat. no. EA88.1). Safe-Lock Tubes 1.5 ml (Eppendorf, cat. no ) mm, GP Express Plus Membrane (Millipore, cat. no. SCGPT02RE). Lint-free Kimwipes (Kimberly-Clark, cat. no. 7551). Sponge AS 60 (Astrein). Blossom Nitril Examination Gloves (Mexpo International, cat. no. BM NPF-GAV). Pipette tip, yellow, 200 ml (Greiner, cat. no ). Pipetman P1000/P200/P20/P2 (Gilson). 30 ml Tall Tip Pipette Tips (Matrix/Apogent Discoveries, cat. no. 7631). Matrix Impact2 12-Channel Pipettor, ml (Matrix/Apogent Discoveries, cat. no. 2019). Spatule (Bochem, cat. no. 3622). Sekuroka-mask (Carl Roth, cat. no ). Sekuroka-safety glasses (Carl Roth, cat. no. Y254.1). Analytical balance APX-200 (Denver Instrument). 384 MALDI target plate, stainless steel (Bruker Daltonics, cat. no ). MTP target frame III (Bruker Daltonics, cat. no ). Thermo-hygrometer WS 9400 (Conrad, cat. no ). Minishaker Model MS 1 (IKA). Ultrasonic cleaner (Branson, Model B2510-MT), including a solid insert tray. Incubator (Memmert, model 200). Forma/Thermo 420 Incubator Orbital Tabletop Shaker. Analytical balance APX-200 (Denver Instrument). Centrifuge 5804R (Eppendorf, cat. no ). Fixed angle rotor F /2 ml, including aluminium lid (Eppendorf). Ultraflex I MALDI-TOF mass spectrometer (Bruker Daltonics). FlexControl software 3.0 (Bruker Daltonics). FlexAnalysis software 2.4 (Bruker Daltonics). MALDI Biotyper software 1.1/2.0 (Bruker Daltonics). ClinProTools 2.1/2.2 software (Bruker Daltonics) m CRITICAL In our laboratory, we use MALDI mass spectrometers and supporting data accumulation software from Bruker Daltonics. However, the method is restricted not only to the equipment and software of this company. Although we cannot include all alternatives, we would like to mention that the protocol for preparation of bacterial samples can also be applied using other MALDI-TOF mass spectrometers from well-known companies, e.g., Applied Biosystems, Waters and Shimadzu. For efficiently adapting the protocol described here, it is important to consider the basic mass spectrometric values described in the text and to adopt the protocol with the respective data accumulation software of the specific mass spectrometer applied. As software for generation of bacterial reference mass spectra libraries, as well as for the classification and identification of bacteria, we use the MALDI Biotyper. To implement the method described in this protocol, the MALDI Biotyper can be tested on the basis of a month s free trial. Alternative software from commercial or academic sources as described in Box 1 can also be applied. REAGENT SETUP. LB (Luria-Bertani) medium (10 g tryptone/peptone, 5 g yeast extract, 10 g NaCl add to 1 liter with double distilled water). Autoclave and filter through a 0.2-mm nitrocellulose filter to remove particles. Autoclaved mixture can be stored at room temperature ( C) until use.. 40% (wt/vol) glucose solution (40 g D-(+)-glucose anhydrous add to 100 ml with double distilled water). Sterile the filtrate (0.22 mm) solution and store at room temperature.. LB-glucose (LB medium + 1% (vol/vol) glucose). Under sterile conditions, add 12.5 ml 40 % (wt/vol) glucose solution to 500 ml autoclaved and filtrated (0.22 mm) LB media.. LB agar (10 g tryptone/peptone, 5 g yeast extract, 10 g NaCl, 15 g agar add to 1 liter with double distilled water, ph 7.5). Autoclaved mixture can be stored at room temperature. m CRITICAL Fresh liquid media or autoclaved liquid media can be stored at room temperature until use (several weeks). For plating, heat up LB agar until dissolving, cool down to 50 1C and plate in Petri dish. Store the agar plates in a refrigerator at 8 1C up to a month. 736 VOL.4 NO NATURE PROTOCOLS

6 PROCEDURE Cell culture 1 Cell culture can be performed either on solid medium (option A) or in liquid medium (option B). (A) Cell culturing on solid medium TIMING B6 48 h (i) For cell culturing of Enterobacteriaceae such as E. coli on agar plates, incubate all dilutions for up to 2 days at an optimal temperature (generally between 28 and 37 1C). (ii) Resuspend bacteria from single colonies grown on the solid medium plate in 1 ml double distilled water. m CRITICAL STEP For good quality spectra, only one single colony should be applied. It is very important that the user always compares mass spectra of bacterial samples that were grown under similar conditions, particularly for bacterial analysis on the subspecies level. It is generally important that the bacteria used for comparative mass spectrometric analysis have entered the same growth phase (use samples that are either in the log or in the stationary phase). (iii) Centrifuge this sample in 1.5 ml tubes at 10,000g at room temperature for B2 min and discard the supernatant. (iv) The pellet can be additionally washed with 1 ml double distilled water; discard the supernatant again. However, in many cases, this step is not necessary. Continue with Step 2.! CAUTION Whatever culture conditions are applied, handle all biological samples as a potential source of pathogens. Use appropriate protective attire (lab coats, safety glasses, latex gloves and other conventional measures applied in the microbiology laboratory) and dispose all biohazardous materials properly. (B) Cell culturing in liquid medium TIMING B7 h to overnight (i) Inoculate bacteria (e.g., a colony picked from an agar plate) into 5 ml liquid medium, such as LB-glucose, or other medium that is optimal for the bacteria under investigation. (ii) Incubate for at least 6 h with the possibility of extension to overnight. (iii) Centrifuge 1 ml of the sample in a 1.5-ml tube at 10,000g at room temperature for B2 min and discard the supernatant. (iv) Wash the pellet with 1 ml double distilled water. (v) Centrifuge the sample at 10,000g at room temperature for B2 min and discard the supernatant. (vi) The pellets can be additionally washed with 1 ml double distilled water, centrifuged, and discard the supernatant again.! CAUTION Handle all biological samples as a potential source of pathogens. Use appropriate protective attire (lab coats, safety glasses, latex gloves and other conventional measures applied in the microbiology laboratory) and dispose all biohazardous materials properly. m CRITICAL STEP For good quality mass spectra, a single bacterial colony should be used. Particularly for the identification of bacterial subspecies, it is very important that the user only compares mass spectra of bacterial samples that were grown under similar conditions. Extraction of proteins from bacterial cells 2 The extraction of proteins from whole bacterial cells can be performed using ethanol formic acid extraction (option A) or TFA extraction (option B). (A) Ethanol formic acid extraction TIMING B 30 min (i) To inactivate bacteria, re-suspend a bacterial pellet in 300 ml double distilled water and add 900 ml ethanol at room temperature ( C).! CAUTION In many cases, inactivation is necessary for potentially pathogenic bacteria because of biosafety reasons. To assess bacterial cell viability, apply dilutions to the appropriate media (e.g., agar plates) to screen for potentially surviving cells after a sufficient time of incubation at the appropriate temperature. These conditions depend on the bacteria under investigation. PAUSE POINT Ethanol-inactivated bacteria can be stored several weeks at 4 1C. (ii) Centrifuge the ethanol-inactivated sample at 10,000g at room temperature for B2 min. (iii) Remove the supernatant by decanting and centrifuge again at 10,000g at room temperature for B2 min. (iv) Remove the residual ethanol with a pipette. Allow the sample to dry for 1 min. (v) Add 10 ml of 70% formic acid to the pellet and mix thoroughly by pipetting and/or by vortexing. The pellet should be dissolved as much as possible. (vi) Add 10 ml of pure acetonitrile (the same volume as in Step 2A(v)) and mix carefully. (vii) Centrifuge at 10,000g at room temperature for B2 min. To gain protein samples that are free from residual material in the pellet, transfer the supernatant into a new tube immediately.! CAUTION This protocol is suitable for inactivating bacteria without spore formation. Acetonitrile is highly flammable and toxic. Formic acid is corrosive. Work under fume hood and wear appropriate protective attire (clothing and gloves). NATURE PROTOCOLS VOL.4 NO

7 m CRITICAL STEP Use inert reaction tubes (e.g., from Eppendorf) to avoid dissolving of plastic additives, which can disturb the MALDI process. To improve the sensitivity of the procedure, the user might reduce the volumes applied in Steps 2(v) and (vi) proportionally. PAUSE POINT Supernatant can be stored up to 1weekat 20 1C. (B) TFA extraction TIMING B 45 min (i) Place 5 mg of bacterial sample into a tube. (ii) Add 50 ml of80%tfa.! CAUTION Wear suitable protective clothing and work under fume hood. (iii) Resuspend by pipetting until complete dissolution denaturation of the bacterial sample is achieved. In the presence of spores, the solution will not become clear but will stay turbid. Wait for min. (iv) Add 3 volumes of double distilled water (150 ml). TABLE 1 Cleaning procedure for stainless steel MALDI targets. Step Action 1 Remove target from holder 2 Rinse with acetone and wipe with lint-free tissue 3 Rinse with double distilled water and wipe clean 4 To remove traces, use liquid soap and rub with a sponge over the target but do not scratch it 5 Rinse whole plate for B 2 min with double distilled water 6 Sonicate 5 min in HPLC-grade water (ultrasonic bath must be clean) 7 Rinse with HPLC-grade water 8 Wipe dry and keep overnight in an incubator at 37 1C This rather unusual procedure applies liquid soap to get rid of all residual sticky bacterial material from the target plate. Extensive washing of the target plate in Steps 5 7 is necessary to remove detergents on the plate surface. (v) Add an equal volume of pure acetonitrile (200 ml) and mix by vortexing. (vi) Centrifuge at 10,000g at room temperature for 2 min. A volume of 1 ml of the supernatant is sufficient for the MALDI target preparation in one spot (Step 6).! CAUTION This protocol is suitable for inactivating bacteria with spore formation. Acetonitrile is flammable and toxic. TFA is highly corrosive. Work under fume hood and wear appropriate protective attire (clothing and gloves). To assess bacterial cell viability, apply dilutions to appropriate medium to screen for potentially surviving cells. m CRITICAL STEP Use inert reaction tubes (e.g., from Eppendorf) to avoid dissolving of plastic additives, which can disturb the MALDI process. As for the ethanol formic acid extraction procedure, the sensitivity of the method can be increased by reducing the volumes applied in the Steps 2(ii) and (iv v) proportionally. PAUSE POINT Supernatant can be stored up to 1 week at 20 1C. MALDI target preparation TIMING B1.5 h 3 Use a flat stainless steel MALDI target. The target should be properly cleaned before use. A washing procedure is summarized in Table 1. 4 Prepare a matrix solution; take some tiny crumbles of a-cyano-4-hydroxy-cinnamic acid and determine the weight of this matrix. Prepare a solution of 50% acetonitrile and 2.5% TFA (500 ml acetonitrile, 475 ml double distilled water and 25 ml pure TFA) designated as stock solution. Add the stock solution to the weighted matrix to yield a concentration of 10 mg ml 1 a-cyano-4-hydroxy-cinnamic acid (termed as matrix solution ).! CAUTION Acetonitrile is flammable and toxic. TFA is highly corrosive. Work under fume hood. m CRITICAL STEP Use a fresh matrix solution. Aliquots of the matrix can be stored at 20 1C for several months. 5 For mass spectra calibration, apply at least five times of an E. coli standard sample (Table 2)and/orofanappropriate peptide calibration standard (see REAGENTS) on different positions of a stainless steel MALDI target and allow to dry in air. Overlay with 1 ml of the matrix solution directly after drying. Again allow to dry in air. 6 MALDI targets can be prepared using chemically extracted bacterial protein samples (option A) or using bacterial samples without any prior chemical treatment (option B). (A) Preparation of MALDI targets using chemically extracted bacterial protein samples (i) Spot five times 1 ml of each prepared bacterial sample (from Step 2A(vii) or 2B(vi)) onto a stainless steel MALDI target and allow it to dry in air. TABLE 2 Masses derived from E. coli preparations that are used as standard for mass signal calibration. Ribosomal protein Average mass [M+H] + RL36 4, RS22 5, RS34 5, RS33meth 6, RL32 6, RL30 6, RL35 7, RL29 7, RL31 7, RS21 8, RS15 10, RS19 10, Optionally, a conventional peptide mass calibration standard that covers a similar mass range can be applied. 738 VOL.4 NO NATURE PROTOCOLS

8 (B) Preparation of MALDI targets using bacterial samples without any prior chemical treatment (i) Apply bacterial samples from Step 1 directly onto a MALDI target without any prior chemical treatment, which we termed as direct transfer in INTRODUCTION. To do so, smear a single bacterial colony as a thin film directly on a MALDI-TOF target using a pipette tip or an inoculation loop. The visibility of some bacterial material is enough to be applied on the MALDI target.! CAUTION The direct transfer method is a short and simple protocol, which is in any case suitable for nonpathogenic microorganisms. Contamination of medium should be minimized by accurately picking the bacterial clones only from the solid medium plates. Whether this procedure fulfils the biosafety regulations required for the bacterial species under investigation should be verified by a microbiologist. 7 Overlay with 1 ml of the matrix solution directly after drying. Allow to dry in air.! CAUTION While pipetting the matrix solution under fume hood, wear appropriate protective attire (gloves, glasses and mask). m CRITICAL STEP To prevent any oxidative reaction of bacterial proteins that may lead to mass signal shifting in the spectra, it is important to work at a rapid pace. In particular, after drying off the extracts on the stainless steel target, the matrix solution should be added quickly at room temperature with 20 80% air humidity. Make sure that the MALDI target plate is cleaned following previous preparations. Mass spectrometry measurement TIMING B1 h to overnight 8 Insert the MALDI target plate in the mass spectrometer. First, calibrate the MALDI-TOF instrument with E. coli sample applied in Step 5 and use the average masses listed in Table 2. 9 Start the mass spectrometry measurement in an automatic detection mode (see also Box 2). 10 To generate reference spectra, measure four times on the same spot of the target with different laser geometry (spirals). 11 For identification experiments, measure only once on each spot. m CRITICAL STEP After starting the automatic measurement mode, the user should check the first-spectra accumulations and be sure that the procedure works. For reliable identification, sufficient mass accuracy and resolution are very important. Mass accuracy of 200 p.p.m. is required for stringent software analysis (Box 1). The user should never exceed the mass tolerance beyond 300 p.p.m. In general, signals in a mass range of 7,000 10,000 m/z, which have been baseline subtracted (Convex Hull), should have a resolution that is higher than 600 with a signal-to-noise ratio of 2 and an absolute signal intensity of at least 1,000. PAUSE POINT Automatic measurements of 384 spots are usually performed overnight. Compilation of reference mass spectra TIMING B2 h 12 The compilation of a library of reference mass spectra is required if the bacteria under investigation were not analyzed earlier or from which no reference spectra are available from other sources (e.g., the bacterial mass spectra library mentioned in ref. 14). Before compiling mass spectra, open them with the software of the respective mass spectrometer for examination and eliminate low-quality mass spectra; take at least 20 spectra of good quality. Process these spectra according to the descriptions in Box 1. m CRITICAL STEP The mass spectral data can be compiled in generic formats such as XML or ASCII to generate data that are independent from the hardware and from data accumulation software applied. This can be important for transferring mass spectral data into different analysis tools as described in Box 1. Make sure that the mass spectral data produced using the instrument can be exported for the analysis software of choice. Bacterial classification and identification TIMING B2 h 13 Data analysis of raw mass spectra that are produced by the procedure shown above can be performed with bioinformatics tools (Box 1); for phylogenetic classification, we use hierarchical clustering as a simple and powerful tool, whereas for identification of species, we use pattern-matching algorithms (Box 1). For identifying closely related subspecies, we first apply pattern matching. If we obtain ambiguous results, we additionally apply weighted pattern-matching algorithms that overemphasize informative mass signals selected by the user 14 ; thereby, we can in general easily discriminate subspecies from each other in identification experiments. NATURE PROTOCOLS VOL.4 NO

9 m CRITICAL STEP The power of the data analysis depends on strictly following the criteria set by the laboratory protocol described in the previous steps. Quality control measures as described in Box 3 can help to validate the reproducibility of the whole process from bacterial cell culturing to mass spectrometry detection. TIMING The timing depends on the number of samples analyzed. Here the timing refers to one single bacterial sample. Step 1, cell culturing, depends on the culturing conditions: B6 48 h Step 2, chemical extraction of bacterial proteins: B1 45 min Steps 3 7, target preparation: B10 min Steps 8 11, mass spectrometry measurement: B10 min Steps 12 and 13, data analysis: B5 min Troubleshooting advice can be found in Table 3. TABLE 3 Troubleshooting table. Step Problem Possible Reason Solution 4, 7 Poor (inhomogeneous) crystallization Matrix solution; air temperature and/or humidity Prepare fresh matrix solution; check temperature and air humidity if available use an appropriate incubator 6 Smeared sample and matrix Insufficient cleaning and drying of the target 9 Automated measurement is not working MALDI method written in an appropriate file (e.g., AutoXecute.txt file) does not exist 9 11 Salt adducts detected by mass spectrometry Different reference and experimental spectra of the same bacterial subspecies Insufficient rinsing of the target; solvents contain salts (i) Different growth media used (ii) Samples were harvested in different growth phases Follow the cleaning protocol, rinse extensively with double distilled water and afterward with HPLC-grade water, and let dry overnight at 37 1C inan incubator Correct the MALDI method file name and save again Rinse extensively with double distilled water and afterward with HPLC-grade water. Prepare solvents with doubledistilled water Grow bacteria in the same media. Verify that bacteria are collected during the same growth (log or stationary) phase. Eliminate noticeable media-depending peaks using appropriate data analysis software Low-quality mass spectra Low number of bacterial cells Culture cells once again, resuspend bacteria in less volume for chemical treatment, e.g., 5 mlof70% formicacid and 5 ml of acetonitrile Some bacteria are difficult to measure, e.g., bacterial species that produce slurry Use of poor quality tubes containing additives that disturb matrix crystallization Grow a new batch and try again, change the media to reduce the production of slurry Use MS-proved tubes and tips (e.g., from Eppendorf) (continued) 740 VOL.4 NO NATURE PROTOCOLS

10 TABLE 3 Troubleshooting table (continued). Step Problem Possible Reason Solution Low signal-to-noise ratio/low-signal intensity Laser power is set too low PROTOCOL Increase laser power by 1% increments and increase intensity threshold, accumulate more than the usual 1,000 laser shots but check if signal-to-noise ratio is OK Mass peak resolution too low Laser power is set too high Find lowest possible laser power so that the mass signal just emerges out of the background; set mass resolution at 600 Mass spectra accumulation less than 1,000 laser shots 13 Identification of experimental samples with standards show ambiguous results Identification experiments generate poor results Identification using software produces poor results ANTICIPATED RESULTS Using the preparation and mass spectrometry protocol described herein, we detected protein mass patterns in the range of 2,000 20,000 Da. However, for classification and identification analysis, only the mass range from 3,000 to 15,000 Da is important (Fig. 2). The mass spectral data of bacterial species and subspecies can be clustered hierarchically as exemplarily shown in Figure 3. Table 4 shows an identification experiment using pattern-matching algorithms as described in Box 1. The method may not in all cases sufficiently distinguish between very closely related subspecies as might be possible with 16S rrna sequencing. As we have shown recently, the combination of weighted mass pattern-matching analysis and mass spectrometry-based genetic analysis can determine bacterial subspecies and strains very accurately 14,19. The average mass accuracy ( p.p.m.) and the (corresponding) resolution of more than 600 required for pattern-matching analysis (Box 1) are easily achievable in Instrument parameters are set too strictly (e.g., mass resolution and/or signal intensity threshold are set too high) Biological samples usually contain a mixture of bacteria Calibration between reference and experimental spectra are different Calibration between reference and experimental spectra are different 1, Distance level Find appropriate laser power so that the mass signal emerges out of the background, or lower mass signal intensity threshold Apply a complex sample on solid medium and pick a single colony for subsequent processing Perform identification with 300 p.p.m. mass accuracy instead of the usual 200 p.p.m. However, this increases the higher probability of false-positive results Recalibrate the MALDI-TOF instrument with E. coli samples or recalibrate with appropriate data analysis software spectra of bacterial samples by using the E. coli mass standard Pectobacterium carotovorum ssp odoriferum LMG 5863 Pectobacterium betavasculorum DSM Brenneria salicis CFBP 802 Brenneria rubrifaciens CFBP 3619 Erwinia billingiae 661 NCPPB 2285 Erwinia tasmianiensis 1/99 DSM Erwinia pyrifoliae 16/96 DSM Erwinia amylovora CFBP 1232 Pantoea agglomerans LMG 2764 Pantoea agglomerans EhC9-1 Escherichia coli W3350 Escherichia coli 1100 Pantoea dispersa NCPPB 2285 Pantoea dispersa DSM 30073T Figure 3 Phylogenetic classification of a number of bacteria. More information on the underlying mass spectral data handling can be found in Box 1. NATURE PROTOCOLS VOL.4 NO