Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

The protocol demonstrates a rapid method for identification of bacterial proteins using antibiotic induction and mass spectrometry. The main advantage of the technique is it's speed and simplicity. Sample preparation is uncomplicated.

The technique can be extended to the characterization of any pathogenic bacteria, such as virulence factors or antibiotic resistance to better inform appropriate treatment for a bacterial infection. To begin, use a sterile one microliter loop to harvest bacteria from single colonies grown on LB agar plate. And transfer to a two milliliter O-ring ring line screw cap microcentrifuge tube containing 300 microliters of HPLC-grade water.

Then briefly vortex the tube and pellet the cells by centrifugation, at a 0.75 microliter relic water of the sample supernatant on the Stainless Steel MALDI Target, and allow it to dry. Overlay the dried sample spot with 0.75 microliters of a saturated solution of sinapinic acid prepared in 33%acetonitrile, 67%water, and 0.2%trifluoroacetic acid, and allow the spot to dry. Once the spot is dried, load the target in the mass spectrometer.

Open the acquisition software and click MS Linear Mode Acquisition Or mass-to-charge range and mass-to-charge of the lower and upper bounds into their respective fields. Click on the sample spot to be analyzed on the MALDI target template. Then depress the left mouse button, and drag the cursor over the sample spot to specify the rectangular region to be sampled for a laser ablation or ionization.

Release the mouse button to initiate the acquisition and collect 1000 laser shots for each sample spot. If ions are not detected increase the laser intensity by adjusting the sliding scale bar under laser intensity until the protein ion signal is detected. After completion of the MS Linear Mode Acquisition, click the MS/MS Reflectron Mode Acquisition.

Enter the precursor mass to be analyzed into the Precursor Mass field. Next, enter an isolation width in daltons in the Precursor Mass window for the low and high mass side of the precursor mass. Click the CID Off button.

Then click the Metastable Suppressor On button. Adjust the laser intensity to at least 90%of its maximum value by adjusting the sliding scale bar as demonstrated. Click on the sample spot to be analyzed on the MALDI target template, then depress the left mouse button, and drag the cursor over the sample spot to specify the rectangular region to be sampled for laser ablation or ionization, release the mouse button to initiate the acquisition and collect 10, 000 laser shots for each sample spot as demonstrated.

Double-click on the Protein Biomarker Seeker Executable File, and the graphical user interface window will appear. Enter the mass of the protein biomarker in the Mature Protein Mass field, and the mass measurement error in the Mass Tolerance field. Optionally, click on the Complimentary b/y ion Protein Mass Calculator button to calculate the protein mass from a putative complimentary fragment ion pair, and the pop-up window of the Protein Mass Calculator Tool will appear.

Enter the mass to charge ratio of the putative complimentary fragment ion pair, click on the Add Pair button, and the Calculator Protein Mass will appear. Copy paste this value in the Mature Protein Mass field, and close the Protein Mass Calculator tool window. Click on the Set residue Restriction box.

Select the end terminal signal peptide length, and the pop-up with a sliding scale and cursor will appear and move the cursor to the desired signal peptide length. If the signal peptide length is not selected, an unrestricted sequence truncation will be performed by the software. Under the Fragment Ion Condition, select the Residues For Polypeptide Backbone Cleavage by clicking on the boxes of one or more residues, D-E-N, and/or P.And then click on the Enter Fragment Ions Plus One to be search button, and the pop-up fragment page will appear.

Next click on the Add Fragment Ion button, and a dropdown field will appear for each fragment ion to be entered. Enter the mass by charge ratios of the fragment ions and their associated mass by charge tolerance. Then click on the Save and Close button.

In the right side box of How Many Fragment Ions Need To Be Matched, scroll to select the minimum number of fragment ions that must be matched for identification, and select the cysteine residues in their oxidized state. If the proteins are not identified after the search, repeat the search with cysteines in their reduced state. Under the File Setup, click on the Select FASTA file icon to browse and select the FASTA file containing the in silico protein sequences of the bacterial strain previously constructed.

Then select an output folder and create an output file name. Click on the Run Search on File Entries icon. A popup window will appear entitled Confirm Search Parameters displaying the search parameters before the search is initiated.

And if the search parameters are correct, click Begin Search. If the parameters are incorrect, click Cancel and re-enter the correct parameters. Once the search is initiated, the parameter window will close and a new pop-up window with a progress bar will appear, showing the progress of the search and a running tally of the number of identifications found.

Upon completion of the search, the progress bar will be closed automatically, and a summary of the search will be displayed in the log field of the graphical user interface along with a new pop-up window displaying the protein identifications found. In the current study, bacterial cultures were grown in LB with two different concentrations of mytomycin-C. The mass spectrum of bacterial culture grown with a mytomycin-C concentration.

Cold shock protein C, cold shock protein E, and a plasmid-borne protein of unknown function were identified. The unknown protein ion at 9780 was analyzed by MS/MS, And the precursor ion was isolated with a time ion selector window of approximately 100 daltons. The fragment ion at mass to charge 9675.9 is spillover from the dissociation of the metastable protein ion at 9655.

The sequence of the immunity protein for colicin E3 contains basic residue possible sites of ionization. The fragment ions detected from polypeptide backbone cleavage, when the reduced form of cysteine was used during the search. The N-terminal methionine is removed as a post translation modification.

When the bacterial culture was grown at a high mytomycin-C concentration, the immunity protein of bacteriocin was identified. The unknown peak at 9651 was analyzed by MS/MS, and the precursor ion was isolated with a narrower and asymmetric TIS window of minus 75 plus 60 daltons. The sequence of the immunity protein of bacteriocin contains basic residues possible sites of ionization.

The fragment ions detected from polypeptide backbone cleavage, when the reduced form of cysteine was used during the search. with the different concentrations of antibiotics, relative protein abundance may vary. And these were analyzed using mass spectrometry.

When harvesting bacterial cells, observe colony morphology and bacterial growth with respect to antibiotics and concentration. A one microliter loop of cells is necessary to detect proteins.