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

The overall goal of this mass spectrometric procedure, called MSPP or Mass Spectrometry-based PhyloProteomics, is to sub-type bacteria at the strain level. This method can be used to answer key questions in the fields of Microbiology, Epidemiology and Hygiene. For example, it can be used to analyze outbreak situations by visualizing phyloprotomic relatedness.

Compared to other typing methods, MSPP is cheap per sample, and very fast. Nosocomial infections are an issue of increasing importance, and we hope that our MSPP scheme can be extended towards epidemiologic typing of other bacteria, or even fungi. Our MSPP scheme provides insight into relatedness of strains, so for the prediction of relevant phenotypes, such as drug resistance, or even virulence, it does make sense to apply it where these phenotypes correlate with specific genotypes.

To begin this procedure, prepare two tubes of matrix solution by dissolving 2.5 milligrams of HCCA in 250 microliters of an organic solvent mixture, a 50%isonitrile. Add recombinant human insulin as an internal calibrate to one of them, then 47.5%double distilled water and 2.5%trifluoroacetic acid. Next, spread a pinhead sized amount of a colony from the reference strain, directly onto a maldi target plate.

For determination of the exact mass of the calibration peak, place on microliter of test standard on another spot. Overlay each spot of bacteria and standard with 1 microliter of HCCA spiked matrix solution and leave them to crystallize at room temperature. For each strain to be analyzed, repeat the smear preparation.

Overlay each spot with 1 microliter of spiked HCCA matrix solution and leave to crystallize at room temperature. Alternatively, proteins can be extracted from difficult to lie cells. For this, harvest approximately 5 colonies from an Agar plate culture with an inoculation loop.

Suspend them in 300 microliters of double distilled water in a 1.5 milliliter reaction tube, and mix them thoroughly by repeated pipetting until the bacterial colonies are completely suspended. Then add 900 microliters of absolute ethanol and mix. Centrifuge the sample at 13, 000 g's for 1 minute.

After drying the pellet, re suspend it by pipetting up and down in 50 microliters of 70%formic acid. Then, add 50 microliters of aceto-nitrile and mix. Remove the debris by centrifugation at 13, 000 g's for 2 minutes.

When finished, transfer 1 microliter of the supernamed onto a sample position on a maldi target plate. After allowing the sample to dry, overlay each spot with one microliter of HCCA matrix solution and leave to crystallize at room temperature. At this point, perform MALDI-TOF analysis by gathering 600 spectra in 100 shot steps for each spot.

First, click on the auto execute tab in the configuration software of the mass spectrometer. Open the method by left clicking the method button and choosing the method from the pull down menu. Next, left click the edit button to open the auto execute method editor.

Under the accumulation tab, set the satisfactory shot value to 100 and the sum up value to 600. For each spectrum, choose the calibrant peak from the list, click automatic assign'and press OK'After obtaining mass spectra from each spot, experimentally determine the exact mass of the calibration peak, by opening a spectrum with the spectrum browser and finding the peak at the expected mass as compared to a spectrum without calibrant. Check that the calibrant peak is not obscured by any other biomarker of the organism of interest.

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. Upload file'or copy paste'this protein sequence into the input box of the appropriate bioinformatics resource panel, and compute the theoretical isoelectric point and molecular weight. Copy the results into a spreadsheet with one column containing the gene identifier and another column the molecular weight.

Sort the rows by calculated molecular weight, to facilitate looking up masses. Insert an additional column into the spreadsheet for the molecular weight of the demathianinated form, subtracting 135 dautons from the mono isotopic molecular weight. Assign each major measured biomarker mass to the calculated masses from the reference strain, by looking up the measured mass from the genome table.

In another spreadsheet, record the mass and identifier for each biomarker ion. Now calibrate the mass spectra obtained from the collection isolates as done for the reference strain. For one isolate per row, identify variant biomarkers in the mass spectra, record the measured mass for each biomarker and the predicted isoform in the respective columns.

The MALDI-TOF mass spectrum of C-Jejuni Subspecies doylei ATCC 49349 contains 14 singularly charged biomarker ions that could be identified by comparison of the calculated molecular masses with the reference spectrum. Within the collection, varying iso forms were detected for L32 without methionine, L33, and the hypothetical protein encoded by gil152939117, S20 M, and S15 M.The remaining masses assigned to biomarker ions were invariable in the tested isolates. The amino acid substitution corresponding to the mass shift has been identified by PCR amplification and sanger sequencing of the particular gene using the primers listed here.

The amino acid sequences of all biomarker ions included in the MSPP scheme, as well as the detected alelic isoforms are indicated here. The MLST based UPGMA dendrogram constructed from the examined C jejuni subspecies doylei isolates, shows that each isolate belongs to a different MLST sequence type. And strain LMG7790 has the longest phylogenetic distance.

As seen with the MLST alaysis, isolate LMG7790 has the longest phyloprodeomic distance in the MSPP analysis of the examined C jejuni subspecies doylei isolates. Once mastered, this technique can be done within one day if it's performed properly. While following this procedure, it's important to remember the toxicity of some substances used during the experiment.

For example, aceto-nitrile can cause skin irritation. Additionally, to MSPP other methods like DNA sequencing, can be performed to analyze further parameters, like the presence of resistance genes. The development of this technique paves the way for researchers in the field of microbiology to explore photogeny.

After watching this video, you should have a good understanding of how MSPP works and how it can be applied to specific questions. Don't forget, that working with violent pathogens can be extremely hazardous and precautions such as S2 lab equipment should always be taken.