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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

Abstract

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Introduction

Analysis and identification of undigested proteins by mass spectrometry is referred to as the top-down proteomic analysis1,2,3,4. It is now an established technique that utilizes electrospray ionization (ESI)5 and high-resolution mass analyzers6, and sophisticated dissociation techniques, e.g., electron transfer dissociation (ETD), electron capture dissociation (ECD)7, ultraviolet photo-dissociation (UV-PD)8, etc.

The other soft ionization technique is matrix-assisted laser desorption/ionization (MALDI)9,10,11 that has been less extensively utilized for the top-down analysis, in part because it is primarily coupled to time-of-flight (TOF) mass analyzers, which have limited resolution compared to other mass analyzers. Despite these limitations, MALDI-TOF and MALDI-TOF-TOF instruments have been exploited for the rapid top-down analysis of pure proteins and fractionated and unfractionated mixtures of proteins. For the identification of pure proteins, in-source decay (ISD) is a particularly useful technique because it allows mass spectrometry (MS) analysis of ISD fragment ions, as well as tandem mass spectrometry (MS/MS) of protein ion fragments providing sequence-specific fragment often from the N- and C-termini of the target protein, analogous to Edman sequencing12,13. A drawback to the ISD approach is that, as in Edman sequencing, the sample must contain only one protein. The one protein requirement is due to the need for unambiguous attribution of fragment ions to a precursor ion. If two or more proteins are present in a sample, it may be difficult to assign which fragment ions belong to which precursor ions.

Fragment ion/precursor ion attribution can be addressed using MALDI-TOF-TOF-MS/MS. As with any classical MS/MS experiment, precursor ions are mass-selected/isolated prior to fragmentation, and the fragment ions detected can be attributed to a specific precursor ion. However, the dissociation techniques available for this approach are restricted to primarily high energy collision-induced dissociation (HE-CID)14 or post-source decay (PSD)15,16. HE-CID and PSD are most effective at fragmenting peptides and small proteins, and the sequence coverage can, in some cases, be limited. In addition, PSD results in polypeptide backbone cleavage (PBC) primarily on the C-terminal side of aspartic and glutamic acid residues by a phenomenon called the aspartic acid effect17,18,19,20.

MALDI-TOF-MS has also found a niche application in the taxonomic identification of microorganisms: bacteria21, fungi22, and viruses23. For example, MS spectra are used to identify unknown bacteria by comparison to a reference library of MS spectra of known bacteria using pattern recognition algorithms for comparison. This approach has proved highly successful because of its speed and simplicity, although requiring an overnight culturing of the isolate. The protein ions detected by this approach (usually under 20 kDa) comprise a MS fingerprint allowing taxonomic resolution at the genus and species level and in some cases at the sub-species24 and strain level25,26. However, there remains a need to not only taxonomically classify potentially pathogenic microorganisms but also identify specific virulence factors, toxins, and antimicrobial resistance (AMR) factors. To accomplish this, the mass of peptides, proteins, or small molecules are measured by MS and subsequently isolated and fragmented by MS/MS.

Pathogenic bacteria often carry circular pieces of DNA called plasmids. Plasmids, along with prophages, are a major vector of horizontal gene transfer between bacteria and are responsible for the rapid spread of antimicrobial resistance and other virulence factors across bacteria. Plasmids may also carry antibacterial (AB) genes, e.g., colicin and bacteriocin. When these genes are expressed and the proteins secreted, they act to disable the protein translation machinery of neighboring bacteria occupying the same environmental niche27. However, these bactericidal enzymes can also pose a risk to the host that produced them. In consequence, a gene is co-expressed by the host that specifically inhibits the function of an AB enzyme and is referred to as its immunity protein (Im).

DNA-damaging antibiotics such as mitomycin-C and ciprofloxacin are often used to induce the SOS response in Shiga toxin-producing E. coli (STEC) whose Shiga toxin gene (stx) is found within a prophage genome present in the bacterial genome28. We have used antibiotic induction, MALDI-TOF-TOF-MS/MS, and top-down proteomic analysis previously to detect and identify Stx types and subtypes produced by STEC strains29,30,31,32. In the previous work, STEC O113:H21 strain RM7788 was cultured overnight on agar media supplemented with mitomycin-C. However, instead of detecting the anticipated B-subunit of Stx2a at m/z ~7816, a different protein ion was detected at m/z ~7839 and identified as a plasmid-encoded hypothetical protein of unknown function33. In the current work, we identified two plasmid-encoded AB-Im proteins produced by this strain using antibiotic induction, MALDI-TOF-TOF-MS/MS, and top-down proteomic analysis using standalone software developed to process and scan in silico protein sequences derived from whole-genome sequencing (WGS). In addition, the possibility of post-translation modifications (PTM) involving sequence truncation were incorporated into the software. The immunity proteins were identified using this software from the measured mass of the mature protein ion and sequence-specific fragment ions from PBC caused by the aspartic acid effect and detected by MS/MS-PSD. Finally, a promoter was identified upstream of the AB/Im genes in a plasmid genome that may explain the expression of these genes when this strain is exposed to a DNA-damaging antibiotic. Portions of this work were presented at the National American Chemical Society Fall 2020 Virtual Meeting & Expo (August 17-20, 2020)34.

Protocol

1. Microbiological sample preparation

  1. Inoculate 25 mL of Luria broth (LB) in a 50 mL conical tube with E. coli O113:H21 strain RM7788 (or another bacterial strain) from a glycerol stock using a sterile 1 µL loop. Cap the tube and pre-culture at 37 °C with shaking (200 rpm) for 4 h.
  2. Aliquot 100 µL of pre-cultured broth and spread onto a LB agar plate supplemented with 400 or 800 ng/mL of mitomycin-C. Culture agar plates statically overnight in an incubator at 37 °C.
    CAUTION: STEC strains are pathogenic microorganisms. Perform all microbiological manipulations, beyond culturing, in a BSL-2 biosafety cabinet.
  3. Harvest bacterial cells from single visible colonies using a sterile 1 μL loop and transfer to a 2.0 mL O-ring-lined screw-cap microcentrifuge tube containing 300 μL of HPLC-grade water. Cap the tube, vortex briefly, and centrifuge at 11,337 x g for 2 min to pellet the cells.

2. Mass spectrometry

  1. Spot 0.75 μL aliquot of the sample supernatant onto the stainless steel MALDI target and allow it to dry. Overlay the dried sample spot with a0.75 μL aliquot of a saturated solution of sinapinic acid in 33% acetonitrile, 67% water, and 0.2% trifluoracetic acid. Allow the spot to dry.
  2. Analyze the dried sample spots using a MALDI-TOF-TOF mass spectrometer.
    1. After loading the MALDI target into the mass spectrometer, click the button for MS linear mode acquisition in the acquisition software. Enter the m/z range to be analyzed by entering the m/z of the lower and upper bounds (e.g., 2 kDa to 20 kDa) into their respective fields in the acquisition method software.
    2. Click on the sample spot to be analyzed on the MALDI target template in the software. Then, depress the left mouse button and drag the mouse cursor over the sample spot to specify the rectangular region to be sampled for laser ablation/ionization. Release the mouse button and the acquisition will initiate. Collect 1,000 laser shots for each sample spot.
      NOTE: Data acquisition is displayed in real-time in the software acquisition window.
    3. If no ions are detected, increase the laser intensity by adjusting the Sliding Scale Bar under Laser Intensity in the software until the protein ion signal is detected. This is referred to as threshold.
      NOTE: Prior to the sample spot analysis, externally calibrate the instrument in MS linear mode with protein calibrants whose m/z span the range being analyzed, e.g., the +1 and +2 charge states of protein calibrants: cytochrome-C, lysozyme, and myoglobin cover a mass range of 2 kDa to 20 kDa. An intermediate mass within the specified mass range is used as a focus mass, e.g., 9 kDa. The focus mass is the ion whose m/z is optimally focused for detection by the linear mode detector.
    4. When the MS linear mode acquisition is complete, click the button for MS/MS reflectron mode acquisition in the acquisition software. Enter the precursor mass to be analyzed into the Precursor Mass field. Next, enter an isolation width (in Da) into the Precursor Mass Window for the low and high mass side of the precursor mass, e.g., ±100 Da.
    5. Click on the CID Off button. Click on the Metastable Suppressor ON button. Adjust the laser intensity to at least 90% of its maximum value by adjusting the sliding scale bar under the Laser Intensity in the software.
    6. Click on the sample spot to be analyzed on the MALDI target template in the software. Then depress the left mouse button and drag the mouse cursor over the sample spot to specify the rectangular region to be sampled for laser ablation/ionization. Release the mouse button, and the acquisition will initiate. Collect 10,000 laser shots for each sample spot.
      NOTE: Prior to the sample spot analysis, the instrument should be externally calibrated in MS/MS-reflectron mode using the fragment ions from post-source decay (PSD) of the +1 charge state of alkylated thioredoxin35.
  3. Do not process raw MS data. Process MS/MS-PSD raw data using the following sequence of steps in the specified order: advanced baseline correction (32, 0.5, 0.0) followed by noise removal (two standard deviations) followed by Gaussian smoothing (31 points).
  4. Manually inspect MS/MS-PSD data for the presence of prominent fragment ions generated by PBC19,20.
  5. Evaluate MS/MS data with respect to the absolute and relative abundance of fragment ions and their signal-to-noise (S/N). Use only the most abundant fragment ions for protein identification, especially if the MS/MS-PSD data is noisy.

3. In silico protein database construction

  1. Generate a text file containing in silico protein sequences of the bacterial strain, which will be scanned by the Protein Biomarker Seeker software for the protein identification. Protein sequences are derived from whole-genome sequencing (WGS) of the strain being analyzed (or a closely related strain).
  2. Access the NCBI/PubMed (https://www.ncbi.nlm.nih.gov/protein/) website to download approximately 5,000 protein sequences of the specific bacterial strain (e.g., Escherichia coli O113:H21 strain RM7788) being analyzed. The maximum download size is 200 sequences.
    1. In consequence, copy and paste the 25 downloads into a single text file. Select the FASTA (text) format for each download.

4. Operating Protein Biomarker Seeker software

  1. Double click on the Protein Biomarker Seeker executable file. A graphical user interface (GUI) window will appear (Figure 1, top panel).
  2. Enter the mass of the protein biomarker (as measured in MS-linear mode) into the Mature Protein Mass field. Next, enter the mass measurement error into the Mass Tolerance field. The standard mass measurement error is ±10 Da for a 10,000 Da protein.
  3. Optionally, click on the Complementary b/y ion Protein Mass Calculator button in order to calculate the protein mass from a putative complementary fragment ion pair (CFIP or b/y). A pop-up window, Protein Mass Calculator Tool, will appear (Figure 1, bottom panel).
    1. Enter the m/z of the putative CFIP and click on the Add Pair button. The calculated protein mass will appear.
    2. Copy and paste this number into the Mature Protein Mass field and close the Protein Mass Calculator Tool window.
  4. Select an N-terminal Signal Peptide Length by clicking on the Set Residue Restriction box. A pop-up with a sliding scale and cursor will appear. Move the cursor to the desired signal peptide length (maximum 50). If no signal peptide length is selected, an unrestricted sequence truncation will be performed by the software.
  5. Under the Fragment Ion Condition in the GUI, select residues for polypeptide backbone cleavage (PBC). Click on the boxes of one or more residues: D, E, N, and/or P.
    1. Click on the Enter Fragment Ions (+1) To Be Searched button. A pop-up Fragment Page will appear. Next, click on the Add Fragment Ion button, which corresponds to the number of fragment ions to be entered, i.e., one click for each fragment ion. A dropdown field will appear for each fragment ion to be entered.
    2. Enter the m/z of the fragment ions and their associated m/z tolerance. When completed, click on the Save and Close button.
      NOTE: A reasonable m/z tolerance is ±1.5.
    3. Select the minimum number of fragment ions that must be matched for an identification by scrolling to the desired number in the box to the right of How Many Fragment Ions Need to be Matched.
      NOTE: Three matches should be adequate.
    4. Select cysteine residues to be in their oxidized state by clicking on the corresponding circle. If no protein identifications are found after the search, repeat the search with cysteines in their reduced state. If no identifications are found after the search, widen the fragment ion tolerance to ±3 and repeat the search.
  6. Under the File Setup, click on the Select FASTA File button to browse and select the FASTA (text) file containing the in silico protein sequences of the bacterial strain previously constructed in protocol steps 3.1 to 3.2. Then select an output folder and create an output file name.
  7. Click on the Run Search on File Entries button. A pop-up window will appear entitled Confirm Search Parameters (Figure 1, bottom panel), displaying the search parameters before the search is initiated.
  8. If the search parameters are correct, click on the Begin Search button. If the search parameters are not correct, click on the Cancel button and re-enter the correct parameters. Once the search is initiated, the parameter window closes, and a new pop-up window with a progress bar appears (Figure 1, bottom panel) showing the progress of the search and a running tally of the number of identifications found.
  9. Upon completion of the search (a few seconds), the progress bar automatically closes, and a summary of the search is displayed in the Log field of the GUI (Figure 2, top panel). In addition, a new pop-up window will also appear displaying the protein identification(s) if any (Figure 2, bottom panel).
    ​NOTE: In silico protein sequences having unrecognized residues, e.g., U or X, are automatically skipped from the analysis and these sequences are subsequently reported with a separate pop-up window to alert the operator as to which (if any) sequences were skipped upon completion of the search.

5. Post-search confirmation of protein sequence

  1. Confirm the correctness of a candidate sequence by manual analysis.
    NOTE: The purpose of the Protein Biomarker Seeker software is to identify a protein sequence with high accuracy by eliminating many obviously incorrect protein sequences from consideration and incorporating sequence truncation as a possible PTM in the mature protein. As the number of possible candidate sequences returned are few, manual confirmation is manageable.
  2. Generate a table of the average m/z of b- and y-type fragment ions of the candidate sequence using any mass spectrometry or proteomic software having such functionality. Compare the average m/z of in silico fragment ions on the C-terminal side of D-, E-, and N-residues (and on the N-terminal side of P-residues) to the m/z of prominent fragment ions from the MS/MS-PSD data.
    NOTE: The most prominent MS/MS-PSD fragment ions should be easily matched to D-, E-, and N-associated in silico fragment ions. However, the aspartic acid effect fragmentation mechanism is less efficient near the N- or C-termini of a protein sequence36.

Results

Figure 3 (top panel) shows the MS of STEC O113:H21 strain RM7788 cultured overnight on LBA supplemented with 400 ng/mL mitomycin-C. Peaks at m/z 7276, 7337, and 7841 had been identified previously as cold-shock protein C (CspC), cold-shock protein E (CspE), and a plasmid-borne protein of unknown function, respectively33. The protein ion at m/z 9780 [M+H]+ was analyzed by MS/MS-PSD as shown in Figure 3 (bottom panel). The p...

Discussion

Protocol considerations
The primary strengths of the current protocol are its speed, simplicity of sample preparation, and use of an instrument that is relatively easy to operate, be trained on, and maintain. Although bottom-up and top-down proteomic analysis by liquid chromatography-ESI-HR-MS are ubiquitous and far superior in many respects to top-down by MALDI-TOF-TOF, they require more time, labor, and expertise. Instrument complexity can often affect whether certain instrument platforms are lik...

Disclosures

The authors have no conflicts of interest.

Acknowledgements

Protein Biomarker Seeker software is freely available (at no cost) by contacting Clifton K. Fagerquist at clifton.fagerquist@usda.gov. We wish to acknowledge support of this research by ARS, USDA, CRIS grant: 2030-42000-051-00-D.

Materials

NameCompanyCatalog NumberComments
4000 Series Explorer softwareAB SciexVersion 3.5.3
4800 Plus MALDI TOF/TOF AnalyzerAB Sciex
Acetonitrile Optima LC/MS gradeFisher ChemicalA996-1
BSL-2 biohazard cabinetThe Baker CompanySG403A-HE
Cytochrome-CSigmaC2867-10MG
Data Explorer softwareAB SciexVersion 4.9
Focus Protein Reduction-Alkylation kitG-Biosciences786-231
GPMAW softwareLighthouse DataVersion 10.0
IncubatorVWR9120973
LB AgarInvitrogen22700-025
Luria BrothInvitrogen12795-027
LysozymeSigmaL4919-1G
Microcentrifuge Tubes, 2 mL, screw-cap, O-ringFisher Scientific02-681-343
MiniSpin Plus CentrifugeEppendorf22620207
Mitomycin-C (from streptomyces)Sigma-AldrichM0440-5MG
MyoglobinSigmaM5696-100MG
Shaker MaxQ 420HP Model 420Thermo ScientificModel 420
Sinapinic acidThermo Scientific1861580
Sterile 1 uL loopsFisher Scientific22-363-595
Thioredoxin (E. coli, recombinant)SigmaT0910-1MG
Trifluoroacetic acidSigma-Aldrich299537-100G
Water Optima LC/MS gradeFisher ChemicalW6-4

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