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.

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 ...

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

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3.0K Views.  Agricultural Research Service, U.S. Department of Agriculture. 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.

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

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

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 ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...