Name: identification of antibacterial immunity proteins in escherichia coli using maldi-tof-tof-ms/ms and top-down proteomic analysis
Uploaded: 2021-05-23T15:00:00
Description: Watch this Scientific Journal Video about Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis at JoVE.com

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.

1. Microbiological sample preparation
<ol>
	<li>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 &#181;L loop. Cap the tube and pre-culture at 37 &#176;C with shaking (200 rpm) for 4 h.</li>
	<li>Aliquot 100 &#181;L 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 &#176;C. ...

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...

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 &#38; Expo (August 17-20, 2020)34.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4000 Series Explorer software</td>
			<td>AB Sciex</td>
			<td>Version 3.5.3</td>
			<td></td>
		</tr>
		<tr>
			<td>4800 Plus MALDI TOF/TOF Analyzer</td>
			<td>AB Sciex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile Optima LC/MS grade</td>
			<td>Fisher Chemical</td>
			<td>A996-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BSL-2 biohazard cabinet</td>
			<td>The Baker Company</td>
			<td>SG403A-HE</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome-C</td>
			<td>Sigma</td>
			<td>C2867-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Explorer software</td>
			<td>AB Sciex</td>
			<td>Version 4.9</td>
			<td></td>
		</tr>
		<tr>
			<td>Focus Protein Reduction-Alkylation kit</td>
			<td>G-Biosciences</td>
			<td>786-231</td>
			<td></td>
		</tr>
		<tr>
			<td>GPMAW software</td>
			<td>Lighthouse Data</td>
			<td>Version 10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>9120973</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Invitrogen</td>
			<td>22700-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth</td>
			<td>Invitrogen</td>
			<td>12795-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L4919-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, 2 mL, screw-cap, O-ring</td>
			<td>Fisher Scientific</td>
			<td>02-681-343</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniSpin Plus Centrifuge</td>
			<td>Eppendorf</td>
			<td>22620207</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitomycin-C (from streptomyces)</td>
			<td>Sigma-Aldrich</td>
			<td>M0440-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Myoglobin</td>
			<td>Sigma</td>
			<td>M5696-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker MaxQ 420HP Model 420</td>
			<td>Thermo Scientific</td>
			<td>Model 420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sinapinic acid</td>
			<td>Thermo Scientific</td>
			<td>1861580</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 1 uL loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-595</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioredoxin (E. coli, recombinant)</td>
			<td>Sigma</td>
			<td>T0910-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Optima LC/MS grade</td>
			<td>Fisher Chemical</td>
			<td>W6-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of antibacterial immunity proteins in escherichia coli using maldi-tof-tof-ms/ms and top-down proteomic analysis

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.

Figure 3&#160;(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...

Watch this Scientific Journal Video about Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis at JoVE.com

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

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.

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

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

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