Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

1. Prepare a Safe Workplace by Considering Biosafety Conditions

1. Become familiar with the laboratory and safety regulations that are of relevance for working with microorganisms. Most human pathogenic microorganisms must be handled at biosafety level 2 conditions but some, such as Salmonella enterica serovar Typhi, require biosafety level 3. Information on level of handling each pathogen can be accessed at www.cdc.gov/biosafety.
2. Regardless of the biohazard classification of the specific microorganism, regard all materials that came in contact with the infectious agent as infectious waste that must be autoclaved before disposal. Respect regional safety guidelines for hazardous materials and biological substances. Ensure that suitable containers for immediate and proper disposal of potentially contaminated materials (biohazards) are available.
3. Ensure that sterile instruments (inoculation loops), solutions and culture media (agar plates) are available before commencing bacterial culture.
4. Wash hands with antiseptic soap and warm water immediately after handling infectious microorganisms.

2. Select Reference and Collection Isolates

1. Select and obtain one standard genome-sequenced reference isolate along with the sequences of the encoded proteome, ideally in FASTA format. If more genome-sequenced strains are available, include these in the analysis.
   Note: This isolate/these isolates will later on be used to predict the identity of the mass peaks observed in mass spectrometry (see section 7).
2. Select and obtain a variety of potentially diverse isolates in such a way that they cover the phylogeny of the species or subspecies of interest.
   Note: These isolates will later on be used to demonstrate the variability of biomarkers in the population (see section 8).
3. Ensure that the entire collection and reference isolate(s) are properly typed by the respective gold standard for this particular organism^18–20.
   Note: This may include a variety of (sub)-typing methods, but will likely resort to MLST, which still is the standard method to demonstrate genetic diversity of most microbial species.
4. To infer the phylogeny within the collection, calculate a phylogram from the typing data, e.g., using the unweighted pair group method with arithmetic mean (UPGMA) in MEGA6 software for MLST data²¹. For MLST data, also consult a MLST database and assign sequence types and respective clonal complexes²².
   Note: This will later on be used to analyze the congruency of MSPP with the earlier gold standard typing method (see section 9).

3. Prepare a MALDI Target Plate

CAUTION: TFA is a strong acid. Improper use of TFA bears the risk of severe skin burns, eye damage and severe irritation of the upper respiratory tract if inhaled. Therefore, stringent safety measures must be respected and proper personal protective equipment (PPE) including safety goggles, face shields, appropriate gloves, boots, or even a full protective suit is needed, while handling TFA. Possible exposure to TFA must be controlled by handling the substance under adequate ventilation with an effective exhaust ventilation system. In case of insufficient ventilation, a respirator with approved filter must be used. Additionally, TFA is harmful to aquatic life with long lasting effects. Any release of TFA in waste water to the environment must be avoided.

Note: Before spotting the samples onto a MALDI target, clean the target plate thoroughly if the plate was used previously.

1. Prepare 100 ml 70% aqueous ethanol solution using 30 ml deionized water and 70 ml pure ethanol.
2. Prepare 250 µl of an 80% aqueous trifluoroacetic acid (TFA) solution by mixing 50 µl of deionized water and 200 µl 100% TFA in a reaction tube and vortexing the tube for 1 min.
3. Clean the MALDI target by putting it into a glass dish and submersing it in 70% aqueous ethanol for about 5 min at room temperature.
4. Rinse the target under hot water.
5. Using a paper tissue, wipe the target plate intensively with 70% aqueous ethanol solution to remove all previous samples and other potential debris.
6. If further cleaning is required, rinse under hot water while wiping with a paper tissue.
7. Remove residual, and potentially invisible, contaminants, by covering the target surface with a thin layer of 80% aqueous TFA (~100 µl per 96 spots) and wiping all target positions clean with a paper tissue.
8. Finally, rinse the target to remove acid, wipe it dry using a paper tissue, and leave it for at least 15 min at room temperature to evaporate residual liquid.

4. Preparation of an α-Cyano-4-hydroxy-cinnamic Acid Matrix Solution Containing an Internal Calibrant

1. Prepare a saturated matrix solution by dissolving 10 mg α-cyano-4-hydroxy-cinnamic acid (HCCA) in 1 ml of a mixture of 50% acetonitrile, 47.5% water, and 2.5% TFA. Residual undissolved HCCA will remain if the solution is saturated.
2. Add recombinant human insulin as an internal calibrant. For this, prepare a stock solution to a final concentration of 10 pg/µl in 50% aqueous acetonitrile, aliquot and store at -20 °C for further use.

5. MALDI-TOF Mass Spectrometry

Note: Culture conditions specific for the organisms of interest must be used. Samples for MALDI-TOF MS can be prepared either by smear preparation or extraction, depending on the organism (see section 8.4.1). While the ethanol-formic acid extraction method provides sufficient inactivation of pathogens, smear preparation has to be performed under sufficient biosafety conditions as required (see section 1). Usually, there is no risk of infection after the application of the matrix, but for specific pathogens specific inactivation protocols are required. Thus, for example MALDI-TOF MS of Nocardia species requires previous lysis of the bacteria in boiling water, following by ethanol precipitation of proteins²³. EI Khéchine et al. developed a procedure for inactivation of Mycobacteria, heating the bacterial colonies at 95 °C for 1 hr in screw-cap tubes containing water and 0.5% Tween 20²⁴.

1. Smear Preparation
   1. Spread a pinhead-sized amount of a bacterial colony directly onto a MALDI target plate position ('spot').
   2. Overlay each spot with 1 µl of HCCA regular matrix or matrix containing the internal calibrant and leave to crystalize at room temperature. For determination of the exact mass of the calibration peak, overlay a control spot with 1 µl Test Standard and 1 µl of HCCA matrix containing the internal calibrant.
      Note: Here, as Test Standard an extract of Escherichia coli DH5 alpha is used that demonstrates a characteristic protein fingerprint in MALDI-TOF MS. It is spiked it with two proteins that extend the upper limit of the detectable mass range.
2. Extraction Method
   1. Harvest approximately five colonies from an agar plate culture with an inoculation loop and thoroughly suspend in 300 µl double-distilled water in a 1.5 ml reaction tube. Add 900 µl absolute ethanol and mix thoroughly by repeated pipetting until the bacterial colonies are completely suspended.
      Note: At this step it is possible and well established to store the samples at -20 °C. Additionally, inactivation of pathogens can be tested by streaking 1-10 µl of the extract onto a suitable agar plate following incubation at optimal growth conditions. Successful inactivation is indicated by the absence of microbial growth.
   2. Centrifuge the sample at 13,000 x g for 1 min, discard the supernatant, and dry the pellet at room temperature for 10 minutes. Resuspend the pellet thoroughly by pipetting up-and-down in 50 µl of 70% formic acid.
   3. Add 50 µl of acetonitrile and mix. Remove debris by centrifugation at 13,000 x g for 2 min. Transfer 1 µl of supernatant onto a sample position on a MALDI target plate and leave to dry for 5 min at room temperature.
   4. Overlay each spot with 1 µl of HCCA matrix containing the internal calibrant and leave to crystalize at room temperature.
3. Recording of Mass Spectra
   Note: Peak-picking from mass spectra is done using the standard procedures recommended (Centroid algorithm; S/N ratio: 2; rel. Intensity threshold: 2%; peak width 3 m/z, baseline subtraction: TopHat)
   1. Calibrate the instrument according to the manufacturers' protocol.
   2. For each spot, gather 600 spectra in 100-shots steps.
      1. Go to the "AutoXecute" tab of the configuration software of the mass spectrometer. Open the "Method" by left-clicking onto the "Method" button and choosing the method e.g., "MBT_AutoX" from the pulldown menu.
      2. Left-click the "Edit…" button right of the "Method" menu to open the "AutoXecute Method Editor". Go to the "Accumulation" tab. Set the "Sum up" value to "600" and the "satisfactory shots in" _x_ "shot steps" value to "100".
4. Internal Spectrum Calibration Procedure
   Note: Minute measurement errors are inherent to mass spectrometry. Depending on intermittent instrument use, instrument temperature and re-calibration, the obtained measurement values may vary between experiments. Following pre-measurement instrument calibration and post-measurement spectrum calibration to an internal calibrant is the most precise way to ensure inter-spectrum comparability.
   1. Perform the following procedures for each calibration peak list:
      1. Start spectrum browser (e.g., flexAnalysis and open spectrum: menu "File"→"Open…".)
      2. Create mass control list with calibrant peak: menu "Method"→"Open…".
      3. Choose method: MBT_Standard.FAMSMethod →"Open".
      4. Edit Mass Control List: Uncheck all Calibrants.
      5. Add Calibrant peak at bottom: Peak Label: "Insulin_HIStag[M+H]+_avg"; m_z: "5808.29"; Tolerance[ppm]: "50"; Check Calibrant checkbox.
      6. Save as, e.g., "MSPP calibrant list".
   2. For each spectrum, choose the calibrant peak from the list, click "Automatic Assign" and press "Ok".

6. Verify the Internal Calibration Procedure

1. Experimentally Determine the Exact Mass of the Calibration Peak.
   1. Prepare two spots with 1 µl Test Standard each (step 5.1.1). Overlay the first with 1 µl regular HCCA matrix, the second with 1 µl calibrant-spiked matrix.
   2. Obtain mass spectra from each spot (section 5.3), and internally calibrate to the Test Standard peaks (section 5.4).
   3. Overlay both spectra by opening them with the spectrum browser (e.g., flexAnalysis and open spectrum: menu "File"→"Open…") and finding the peak at the expected mass (insulin m/z = 5,808.29), which should be present in the calibrant-spiked spectrum, but not in the spectrum obtained with the regular matrix.
2. Check that the Calibrant Peak is not Obscured by Any Other Biomarker of the Organism of Interest.
   1. Prepare two spots (section 5.1) with the reference strain and overlay the first with 1 µl regular matrix, the second with 1 µl calibrant-spiked matrix.
   2. Acquire mass spectra from both spots (section 5.3) and overlay the resulting spectra by opening them with the spectrum browser (e.g., flexAnalysis and open spectrum: menu "File"→"Open…"). Ensure that the calibrant peak is clearly visible in the spectrum obtained with the spiked matrix and not obscured by another adjacent signal. If this is not the case, choose another calibrant for this particular organism.
      Note: Using a spiked internal calibrant significantly increases the precision to determine variations of biomarker masses. Using this method, mass differences down to 1 Da can be detected. Alternatively, also invariant masses originating from the organisms may be used as calibrants. However, by definition, all organism-derived masses must be considered potentially variable, unless proven otherwise.

7. Identify Biomarker Ions in the Reference Strain

1. Measure the Mass Spectrum of the Reference Strain, Using Matrix Spiked with the Internal Calibrant.
   1. Spread a pinhead-sized amount of a bacterial colony (section 5.1) or 1 µl of bacterial protein extract (section 5.2) directly onto a MALDI target plate position ('spot').
   2. Overlay each spot with 1 µl of HCCA matrix containing the internal calibrant and leave the target plate to crystalize at room temperature (section 5.1.2/5.2.4).
   3. Record mass spectra of the reference strain (section 5.3).
2. Internally calibrate the reference spectrum to the calibrant mass (here: insulin at m/z = 5,806.29), and subsequently pre-process by baseline subtraction (TopHat) and smoothing (parameters: SavitzkyGolay; width: 2 m/z, 10 cycles).
   1. Start spectrum browser (e.g., flexAnalysis and open spectrum: menu "File"→"Open…".)
   2. Choose method: pulldown menu "Method" →"Open…", Left-click the method of choice, e.g., "MBT_Standard.FAMSMethod"→ "Open".
   3. Calibrate spectrum by choosing "Internal…" from the pulldown menu "Calibrate". A window opens listing the calibrant peak(s) (section 5.4). Left-click the calibrant peak (Insulin) →Left-click "OK". Choose "Process spectra" from the "Process" pulldown menu.
   4. For baseline subtraction activate the spectrum in the spectrum list at the right side. Choose "Subtract Mass Spectrum Baseline" from the "Process" pulldown menu.
   5. To smooth the spectrum, activate the spectrum in the spectrum list at the right side. Choose "Smooth Mass Spectrum Baseline" from the "Process" pulldown menu.
3. From the genome sequencing data of the reference strain, calculate the theoretical monoisotopic molecular weight of each of the encoded proteins by translating the DNA sequence into the corresponding amino acid sequence using a sequence alignment editor. Copy-paste this protein sequence into the input box at the ExPASy Bioinformatics Resource Portal (http://web.expasy.org/compute_pi/). and press "Click here" to compute pI/Mw. In the case of C. jejuni ssp. doylei calculate the mass of 14 detectable biomarkers.
4. Copy the results into a spreadsheet, with one column containing the gene identifier and the next the molecular weight. Sort the rows by calculated molecular weight to facilitate easier lookup of the masses. Note: Other columns are optional; functional annotation may be especially useful later for interpretation.
5. Insert a second column into the spreadsheet for the molecular weight of the de-methioninated form, subtracting 135 Da from the monoisotopic molecular weight. Note: This is because some proteins undergo posttranslational modification by proteolytic removal of the N-terminal methionine.
6. Assign each major measured biomarker mass to the calculated masses from the reference strain by looking up the measured mass from the genome table prepared above (Table 1).
7. If the biomarker ions cannot be assigned to predicted gene products, consider other posttranslational modifications (methylation, acetylation, prenylation, etc.; see http://www.abrf.org/delta-mass for a compilation of modifications and the associated mass changes). For any other known posttranslational modification that is frequently observed in the organisms of interest add another column to the table and recalculate the molecular weight analogous to the process for the de-methioninated form.
8. Set up another spreadsheet tab, and record for each biomarker ion the mass and identifier in a separate table column.

8. Assess Biomarker Variability in the Population

1. Calibrate mass spectra obtained from collection isolates, as done for the reference strain(s) (section 5.4.2).
2. Identify variant biomarkers in the mass spectra. A variant mass is characterized by the absence of a mass known from the reference spectrum and appearance of a novel mass not present in the reference. The mass difference must conform to a single amino acid exchange (Table 2), or a combination thereof.
3. At one isolate per row, record the measured mass for each biomarker and the predicted isoform in the respective table columns.
   Note: Rarely, some mass shifts may be attributable to several different amino acid exchanges, e.g., both, N exchanged by D and Q exchanged by E, and vice versa, result in a mass shift of 0.985 Da (Table 2). This intrinsic problem cannot be resolved by mass spectrometry alone. Therefore, different isoforms on the protein sequence level having the same mass must be treated as a single MSPP type. Parallel Sanger sequencing of the particular biomarker ion genes confirmed that this problem did not occur while MSPP-typing the C. jejuni ssp. doylei isolate collection used in this study.
4. Confirm novel MSPP types by PCR-amplifying and sequencing the respective biomarker genes. In turn, this also serves as confirmation that the biomarker identity has been assigned correctly.
   1. Culture bacterial isolates under optimal growth conditions. Culture C. jejuni ssp. doylei strains on Columbia agar supplemented with 5% sheep blood at 37 °C under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂). Incubate for ca. 48 hr. Use a separate agar plate for each isolate to avoid cross-contamination.
   2. Extract genomic DNA of the bacterial isolates using an appropriate DNA extraction kit/ automated machinery according to manufacturer's instructions.
   3. Amplify the respective biomarker genes using the primers listed in Table 3. Perform all PCR reactions under the following conditions: denaturation at 94 °C for 30 sec; annealing at 55 °C for 30 sec; elongation at 72 °C for 30 sec.
   4. Determine the DNA sequence of each amplicon by Sanger sequencing using an appropriate amount of genomic DNA (usually 600-700 ng of DNA is sufficient at a concentration of ca. 100 ng/µl) and one of the amplification primers (usually this is done by use of a service provider).
5. In a separate table (see Table 4), record the deduced protein sequence for each novel isoform by using an appropriate translation tool (e.g., Transseq: http://www.ebi.ac.uk/Tools/st/) ²⁵.

9. Calculate a MSPP-based Phylogeny and Compare to the Gold Standard

1. Concatenate the particular biomarker amino acid sequences belonging to the MSPP type of the isolates into one continuous sequence using a sequence alignment editor, such as BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html)²⁶ or the biomarker spreadsheet.
2. Calculate phylogeny by clustering as done for the gold standard typing data, e.g., UPGMA clustering ²¹.
3. Compare the MSPP-based phylogeny to the one obtained with the gold standard^4,13.