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. 
	CAUTION: STEC strains are pathogenic microorganisms. Perform all microbiological manipulations, beyond culturing, in a BSL-2 biosafety cabinet.</li>
	<li>Harvest bacterial cells from single visible colonies using a sterile 1 &#956;L loop and transfer to a 2.0 mL O-ring-lined screw-cap microcentrifuge tube containing 300 &#956;L of HPLC-grade water. Cap the tube, vortex briefly, and centrifuge at 11,337 x g for 2 min to pellet the cells.</li>
</ol>
2. Mass spectrometry
<ol>
	<li>Spot 0.75 &#956;L aliquot of the sample supernatant onto the stainless steel MALDI target and allow it to dry. Overlay the dried sample spot with a0.75&#160;&#956;L aliquot of a&#160;saturated solution of sinapinic acid&#160;in 33% acetonitrile, 67% water, and 0.2% trifluoracetic acid. Allow the spot to dry.</li>
	<li>Analyze the dried sample spots using a MALDI-TOF-TOF mass spectrometer.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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., &#177;100 Da.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>Manually inspect MS/MS-PSD data for the presence of prominent fragment ions generated by PBC19,20.</li>
	<li>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.</li>
</ol>
3.&#160;In silico protein database construction
<ol>
	<li>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).</li>
	<li>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.,&#160;Escherichia coli O113:H21 strain RM7788) being analyzed. The maximum download size is 200 sequences.
	<ol>
		<li>In consequence, copy and paste the 25 downloads into a single text file. Select the FASTA (text) format for each download.</li>
	</ol>
	</li>
</ol>
4. Operating Protein Biomarker Seeker software
<ol>
	<li>Double click on the Protein Biomarker Seeker executable file. A graphical user interface (GUI) window will appear (Figure 1, top panel).</li>
	<li>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 &#177;10 Da for a 10,000 Da protein.</li>
	<li>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).
	<ol>
		<li>Enter the m/z of the putative CFIP and click on the Add Pair button. The calculated protein mass will appear.</li>
		<li>Copy and paste this number into the Mature Protein Mass field and close the Protein Mass Calculator Tool window.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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 &#177;1.5.</li>
		<li>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.</li>
		<li>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 &#177;3 and repeat the search.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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). 
	&#8203;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.</li>
</ol>
5. Post-search confirmation of protein sequence
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>

The authors have no conflicts of interest.

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><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 precursor ion was isolated with a timed-ion selector (TIS) window &#177;100 Da. Fragment ions are identified by their m/z and type/number. The fragment ion at m/z 2675.9 (highlighted with a star) is spillover from the dissociation of the metastable protein ion at m/z 9655 shown in Figure 3 (top panel). The theoretical average m/z of each fragment ion is shown in parentheses based on PBC of the sequence of colicin E3 immunity protein (Im3) shown above. Sites of PBC are highlighted with a red asterisk with the corresponding fragment ion(s) produced. The N-terminal methionine is underlined signifying that it is post-translationally removed in the mature protein. The sequence has a single cysteine residue (boxed) and is therefore considered in its reduced state.
Using the mass of the protein biomarker and a few prominent non-complementary fragment ions: m/z 1813.8, 2128.9, and 4293.7 (&#177;1.5 tolerance) (Figure 1, bottom panel) and restricting PBC to the C-terminal side of D- and E-residues, only one candidate sequence was reported by the software: Im3 protein sequence (without its N-terminal methionine) (Figure 2, bottom panel). When selecting fragment ions for a search, it should be emphasized that any group of non-complementary fragment ions assumes that summing the m/z of any two fragment ions in the group (and subtracting two protons) results in a mass sum that do not fall within the biomarker mass and associated mass tolerance (&#177;10 Da). Draft WGS of RM7788 revealed 5008 protein sequences (open reading frames)37. Of these ~5,000 full protein sequences, 189,490 full and partial sequences (unrestricted truncation) met the biomarker mass criteria (Figure 2, top panel). Those sequences passing the mass criteria then undergo in silico PBC on the C-terminal side of D- and/or E-residues. The resulting fragment ions generated are then compared to the observed fragment ions entered. The candidate sequence reported by the software was based solely on its mass and three D-&#160;and/or&#160;E-specific PBC sites. The specificity achieved by such a small amount of information will be discussed in the next section.
As shown in Figure 3 (bottom panel), the most abundant fragment ions are the result of PBC on the C-terminal side of D- and E-residues via the aspartic acid effect fragmentation mechanism19,20. Two CFIP are observed: b67/y17 (m/z 7645.1 / m/z 2128.9) and b70/y14 (m/z 7959.4 / m/z 1813.8). These CFIP can be used to more accurately calculate the mass of the protein precursor ion using the simple formula: b (m/z) + y (m/z) - 2H+ = protein mass (Da)33. Using the two CFIP, we obtain an average mass of the protein: 9771.6 Da, which is closer to its theoretical value of 9772.5 Da than the measured mass of the protein ion in MS-linear mode: 9779 Da (Figure 3, top panel). Only a few CFIP were detected because most of the precursor ions having the ionizing proton sequestered at the only arginine residue: R80. The higher gas phase basicity of arginine (237.0 kcal/mol38) compared to a lysine residue (K) (221.8 kcal/mol38) is likely responsible for preferential sequestration of the ionizing proton at the only R-residue.
Figure 4 (top panel) shows the MS of STEC O113:H21 strain RM7788 cultured overnight on LBA supplemented with 800 ng/mL mitomycin-C. Figure 4 (top panel) is quite similar to Figure 3 (top panel), although there are differences in the relative abundance of some protein ions due to the differences in antibiotic concentrations utilized. There are also slight shifts in protein biomarker m/z that reflect differences in external calibration of the instrument on different days. Once again, the protein ions at m/z 7272, 7335, and 7838 are CspC, CspE, and a plasmid-borne protein, respectively. In addition, we detect the Im3 protein ion at m/z 9778 (albeit with less abundance than in Figure 3) as well as a protein ion at m/z 9651 [M+H]+. Figure 4 (bottom panel) shows MS/MS-PSD of the protein precursor ion at m/z 9651. The precursor ion was isolated using a narrower and asymmetric TIS window of -75/+60 Da to eliminate contributions of adjacent protein ions at m/z 9539 and 9778. Fragment ions are identified by their m/z and type/number. The sequence of the immunity protein of bacteriocin (Im-Bac) is shown above. Sites of PBC are highlighted with a red asterisk with their corresponding fragment ion(s). The theoretical average m/z of each fragment ion is also shown in parentheses in the spectrum. The Im-Bac sequence also has a single cysteine residue (boxed) and is therefore considered in its reduced state.
Using the protein biomarker mass, three prominent non-complementary fragment ions: m/z 2675.4, 3853.5, and 5772.8 (&#177;1.50 tolerance) from Figure 4 and restricting PBC to only the C-terminal side of D- and/or E- and/or asparagine (N)-residues, only one candidate sequence was reported by the software: Im-Bac protein. The candidate sequence was retrieved after scanning 191,375 full or partial sequences that met the biomarker mass and tolerance (&#177;10 Da) criteria. The candidate sequence was identified by the software-based solely on its mass and three D- and/or E- and/or N-specific PBC sites.
The most prominent fragment ions in Figure 4 (bottom panel) were, once again, the result of PBC on the C-terminal side of D and/or E-residues and also on the N-terminal side of one of the P-residues20. We also observe PBC on the C-terminal side of an N-residues that is also likely to occur by an aspartic acid effect-like fragmentation mechanism39,40. The weakness of the protein precursor ion signal results in a limited number of interpretable fragment ions. The accuracy of the fragment ion m/z declines with fragment ion abundance. No CFIP were detected due presumably to the ionizing proton being sequestered at the only arginine residue (R74) of the protein ion sequence. All fragment ions contain the R74 residue, consistent with this hypothesis.
The promoter of antibacterial immunity genes 
Figure 5&#160;shows a portion of the 6482 bp contig00100 of E. coli strain RM7788 (GenBank: NWVS01000096.1) from whole-genome shotgun sequencing37. The coding regions for colicin E3, its immunity protein (Im3), the immunity protein of bacteriocin (Im-Bac), and a lysis protein are highlighted in yellow. Upstream of the coding region for the colicin E3 gene are the -35 region, the Pribnow box (PB), inverted repeat of the SOS box, the Shine-Dalgarno/ribosomal binding site (SD/RBS)27. There is a nine base-pair intergenic region between colicin E3 and Im3. LexA (a repressor protein and an autopeptidase) binds to the SOS box blocking the expression of genes downstream. Upon DNA damage (e.g., UV radiation or DNA-damaging antibiotics), LexA undergoes self-cleavage allowing expression of genes downstream27,28. Thus, the expression of these two immunity proteins is consistent with exposure of this strain to a DNA-damaging antibiotic.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62577/62577fig01.jpg" /> 
Figure 1: Screen shots of Protein Biomarker Seeker software. Top panel: Graphical user interface (GUI) of the Protein Biomarker Seeker software. Bottom panels: Pop-up windows of Protein Mass Calculator Tool, Fragment Page, Confirm Search Parameters, and Search progress bar. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/62577fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62577/62577fig02.jpg" /> 
Figure 2: Search results of a protein identification using Protein Biomarker Seeker software. Top panel: Summary of search results displayed in the Log Field of the software GUI. Bottom panel: A pop-up window displaying a protein identification using the software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/62577fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62577/62577fig03.jpg" /> 
Figure 3: Mass spectrometry analysis of STEC O113:H21 strain RM7788. Top panel: MS of STEC O113:H21 strain RM7788 cultured overnight on LBA supplemented with 400 ng/mL mitomycin-C. Bottom panel: MS/MS-PSD of the protein precursor ion at m/z 9780 (top panel). The precursor ion was isolated with a TIS window &#177;100 Da. Fragment ions are identified by their m/z and ion type. The sequence of the immunity protein for colicin E3 (Im3) is shown. Basic residues (sites of possible charge sequestration) are highlighted in blue. PBC are highlighted with a red asterisk with the corresponding fragment ion(s) generated. The theoretical average m/z of each fragment ions is shown in parentheses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/62577fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62577/62577fig04.jpg" /> 
Figure 4: Mass spectrometry analysis of STEC O113:H21 strain RM7788. Top panel: MS of STEC O113:H21 strain RM7788 cultured overnight on LBA supplemented with 800 ng/mL mitomycin-C. Bottom panel: MS/MS-PSD of the protein precursor ion at m/z 9651 (top panel). The precursor ion was isolated with an asymmetric TIS window of -75 on the low m/z side of the precursor ion and +60 on the high m/z side of the precursor ion. Fragment ions are identified by their m/z and ion type. The sequence of the immunity protein of bacteriocin (Im-Bac) is shown. Basic residues (sites of possible charge sequestration) are highlighted in blue. PBC are highlighted with a red asterisk with the corresponding fragment ion(s) generated. The theoretical average m/z of each fragment ion is shown in parentheses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/62577fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62577/62577fig05.jpg" /> 
Figure 5: Analysis of a section of the plasmid genome carried by E. coli O113:H21 strain RM7788. A portion of the 6482 bp contig00100 of E. coli O113:H21 strain RM7788 (GenBank: NWVS01000096.1) from whole genome shotgun sequencing37. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/62577fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1 (S1 Im3): Results of benchmarking analysis of software using select fragment ions of Im3 (from Figure 3, bottom panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/S1 Im3 Supplemental File Fagerquist.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 2 (S2 ImBac): Results of benchmarking analysis of software using select fragment ions of Im-Bac (from Figure 4, bottom panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62577/S2 ImBac Supplemental File Fagerquist.pdf" target="_blank">Please click here to download this File.</a>

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 ...

identification-antibacterial-immunity-proteins-escherichia-coli-using

Nanyang Technological University

Research

JoVE Journal

Chemistry

3.1K Views.  Agricultural Research Service, U.S. Department of Agriculture. Het protocol demonstreert een snelle methode voor de identificatie van bacteriële eiwitten met behulp van antibiotica-inductie en massaspectrometrie. Het grote voordeel van de techniek is de snelheid en eenvoud. Monstervoorbereiding is ongecompliceerd.De techniek kan worden uitgebreid tot de karakterisering van pathogene bacteriën, zoals virulentiefactoren of antibioticaresistentie om een geschikte behandeling voor een bacteriële infectie beter te informeren. Gebruik om te beginnen ...

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

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

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

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

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

Genetics

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Immunology and Infection

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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

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