The work was supported by the National Science Foundation (CHE-0749737). The instrument usage was provided by the Mass Spectrometry Facility at the University of the Pacific.

1. Preparation of the Sample Solutions
<ol>
	<li>First prepare the stock solutions of the peptide and the six reference acids using a mixed solvent of methanol and water in a 1:1 volume ratio. The stock solutions should have a concentration of about 10-3 M.</li>
	<li>Weigh out 1 mg of the solid peptide sample, A3CH, in a 1.5 ml Eppendorf tube and add 1.0 ml of mixed solvent of methanol and water, and mix using a vortex.</li>
	<li>Weigh out 1 mg of difluoroacetic acid (DFAH) and add 1.0 ml of the mixed solvent of methanol and water, and mix using a vortex.</li>
	<li>Use the same procedure to make the stock solutions for the other five reference acids chloroacetic acid (MCAH), bromoacetic acid (MBAH), dichloroacetic acid (DCAH), dibromoacetic acid (DBAH), and trifluoroacetic acid (TFAH).</li>
	<li>Draw about 50 &mu;l of the peptide stock solution into a 1.5 ml Eppendorf tube, and draw about 50 &mu;l of the stock solution of DFAH into the same Eppendorf tube. Dilute the mixed solution with 900 &mu;l of the mixed solvent of methanol and water to achieve a final concentration of about 10-4 M. This diluted solution will be used as the sample solution for the mass spectrometry measurements. The actual ratio of the peptide to the reference acid as well as the final concentration of the sample solution will be adjusted based on the ion signal abundances observed in the mass spectrometer.</li>
	<li>Use the same procedure to prepare the sample solutions of the peptide with the other five reference acids.</li>
</ol>
2. Mass Spectrometry Measurement 1: Proton-bound Cluster Ion Formation
<ol>
	<li>The first step of the mass spectrometry measurement is to generate stable proton-bound cluster ions of the peptide with a reference acid.</li>
	<li>Set the instrument in the negative ion MS mode with the ESI needle voltage at -4.5 kV, the capillary voltage at about -35 V, and the drying gas temperature maintained at 150 &deg;C. Set the Q1 peak width and the Q3 peak width both to the calibrated scale (The peak width is an instrument parameter that can be used to adjust the resolution of the peaks. The "calibrated scale" setting allows to display narrow peaks for better peak separation). The capillary voltage and the drying gas temperature can be adjusted to enhance the observed ion abundances.</li>
	<li>Infuse about 0.5 ml of the sample solution of peptide-DFAH into a 1 ml Hamilton syringe and connect the syringe to the ESI needle inlet using PEEK tubing. Then place the syringe onto the syringe pump. Turn on the syringe pump to infuse the sample solution into the ESI needle with the flow rate at 10 &mu;l/min.</li>
	<li>Turn on the ESI needle voltage to activate the ESI process. Turn on the detector. A mass spectrum display should be observed in the profile mode. If the display is in the centroid mode, switch to the profile mode. Watch the proton-bound cluster ion formation by monitoring the peak at m/z 428. The signal abundance of the cluster ion can be adjusted by fine tuning the instrument. One important parameter is the capillary voltage. One can manually change the capillary voltage (typically in the range of -20 to -50 V) to maximize the abundance of the peak at m/z 428.</li>
</ol>
3. Mass Spectrometry Measurement 2: The CID Bracketing Experiments
<ol>
	<li>The next step is to perform the CID bracketing experiment.</li>
	<li>Once the abundance of the cluster ion reaches the desired value (around 100 mV), switch the instrument to the MS/MS mode. In this mode, Q1 functions as a mass filter to isolate the cluster ion, Q2 functions as the collision cell, and Q3 functions as the mass analyzer.</li>
	<li>Set the collision gas (argon, in this case) pressure at 0.5 mTorr and the collision energy at 17 eV. Three peaks should be observed in the mass spectrum display window. The peak at m/z 428 corresponds to the cluster ion, [DFA&bull;H&bull;A3CS]&macr;. The two peaks at m/z 332 and m/z 95 correspond to the deprotonated peptide (A3CS&macr;) and the deprotonated difluoroacetic acid (DFA&macr;), respectively. The minor peak at m/z 298 is a secondary fragment from the deprotonated peptide. Acquire a CID spectrum for 2 min, Figure 3a.</li>
	<li>Perform similar CID experiments and acquire a CID spectrum for the sample solution of the peptide with bromoacetic acid (MBAH), Figure 3b.</li>
	<li>Perform similar CID experiments and acquire the CID spectra for the sample solutions of the peptide with all other reference acids. The resulting CID spectra will be qualitatively similar to Figures 3a and 3b, but the m/z values and the relative peak heights will be different.</li>
</ol>
4. Mass Spectrometry Measurement 3: The Kinetic Method
<ol>
	<li>The last step is to acquire the SRM spectra.</li>
	<li>Switch the spectrum display to centroid and set the instrument to the selected reaction monitoring (SRM) mode. Keep m/z 428 as the isolated ion by the first quadrupole (Q1), and fill in four masses (mass-to-charge ratios) to be monitored by the third quadrupole (Q3). The four masses are m/z 428 (the cluster ion), m/z 332 (the peptide ion), m/z 298 (the fragment of the peptide ion), and m/z 95 (the DFA&macr; ion). Keep the collision gas pressure at 0.5 mTorr.</li>
	<li>Set the collision energy to 11.7 eV and acquire the spectra for 5 min.</li>
	<li>Change the collision energy to 17.6 eV and acquire the spectra for 5 min.</li>
	<li>Change the collision energy to 23.4 eV, and 29.3 eV, and acquire the spectra at both collision energies for 5 min.</li>
	<li>Perform similar measurements for the peptide with all other reference acids.</li>
</ol>
5. Data Analysis
<ol>
	<li>Copy the values of the ion intensities from all the SRM spectra onto an Excel worksheet.</li>
	<li>Calculate the CID product ion branching ratios, ln([A&macr;]/[Ai&macr;]), measured for all six proton-bound clusters at all four collision energies. Sample values are shown in Table 1.</li>
	<li>Plot the values of ln([A&macr;]/[Ai&macr;]) against the values of &Delta;acidHi - &Delta;acidHavg. This will give four plots corresponding to the data at four collision energies, Figure 4a.</li>
	<li>Extract the values of the slopes and the intercepts by linear regression of the four plots. In this case, the slopes are positive values and the intercepts are negative values. Give the symbol "X" to the slopes and the symbol "Y" to the intercepts. The results are shown in Table 2. Multiply the values of Y by -1 and use the symbol Y' to represent the positive values (this allows the y-axis to display positive values). Note, this conversion is optional as long as the corresponding values are used to make the plot for the next step.</li>
	<li>Plot the values of Y' against the values of X, Figure 4b. Linear regression of the plot yields a slope of 1.706 and an intercept of -0.536. The slope corresponds to &Delta;acidH - &Delta;acidHavg. The value of &Delta;acidHavg is known to be 330.5 kcal/mol, which is determined by the set of selected reference acids. The value of the gas-phase acidity of the peptide is then obtained from the slope: &Delta;acidH(A3CH) = 332.2 kcal/mol.</li>
</ol>

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Nothing to disclose.

The successful measurement of the gas-phase acidity of a peptide largely relies on the selection of suitable reference acids. The ideal reference acids are structurally similar organic compounds with well-established gas-phase acidity values. The reference acids should have similar structures to each other. This will ensure a similar entropy of deprotonation for each of the reference acids in the set. The reference acids should have acidity values close to those of the peptides. For shorter cysteine-containing oligopepti...

The acidities of amino acid residues are among the most important thermochemical properties that influence the structures, the reactivity, and the folding-unfolding processes of proteins 9,19. Individual amino acid residues often show different effective acidities depending on their locations in proteins. In particular, the residues located at the active sites often exhibit significantly perturbed acidities. One such example is the cysteine residue residing in the active sites of the thioredoxin super-family of enzymes 20,21. The active site cysteine is unusually acidic compared to those in unfolded proteins 3-5. It has been suggested that the helical conformation may have a significant contribution to the unusual acidity. There are extensive experimental studies on the acid-base properties of peptides carried out in solutions, especially in aqueous solutions 2,6-8. The results were often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects are minimized 9,10.
In order to understand intrinsic acid-base properties of peptides and proteins, it is important to carry out the studies in a solvent-free environment. Here we introduce a mass spectrometry-based method for the determination of the gas-phase acidity. The approach is referred to as the extended Cooks kinetic method. This method has been successfully applied to a wide range of chemical systems for the determination of various thermochemical properties, such as the gas-phase acidity, the proton affinity, the metal ion affinity, the electron affinity, and the ionization energy 11-15,22-26. We have applied this method to determine the gas-phase acidities of a series of oligo cysteine-polyalanine and cysteine-polyglycine peptides 17,18,27. These studies show that the N-terminal cysteine peptides are significantly more acidic than the corresponding C-terminal ones. The high acidities of the former are likely due to the helical conformational effects in which the thiolate anion is strongly stabilized by the interaction with the helix macro-dipole. Because of the non-volatile and thermally labile nature of peptides, the kinetic method is the most practical approach available at present to produce reasonably accurate acid-base thermochemical quantities of peptides 28.
The general scheme and the equation associated with the kinetic method are shown in Figure 1. The determination of the gas-phase acidity of a peptide (AH) starts with the formation of a series of proton-bound cluster anions, [A&bull;H&bull;Ai]&macr; (or [A&macr;&bull;H+&bull;Ai&macr;]&macr;), in the ion source region of the mass spectrometer, where A&macr; and Ai&macr; are the deprotonated forms of the peptide and the reference acids, respectively. The reference acids are organic compounds with known gas-phase acidities. The reference acids should have structures similar to each other (but not necessarily similar to that of the peptide). The similarity of the structures between reference acids ensures the similarity of the entropies of deprotonation among them. The proton-bound cluster anions are mass selected and collisionally activated and subsequently dissociated using collision-induced dissociation (CID) experiments to yield the corresponding monomeric anions, A&macr; and Ai&macr;, with rate constants of k and ki, respectively, shown in Figure 1a. If secondary fragmentations are negligible, the abundance ratio of the CID fragment ions, [A&macr;]/[Ai&macr;], represents an approximate measure of the ratio of the rate constants, k/ki. Under the assumption that there are no reverse activation barriers for both dissociation channels, the CID product ion branching ratios, ln[A&macr;]/[Ai&macr;], will be linearly correlated to the gas-phase acidity of the peptide (&Delta;acidH) and those of the reference acids (&Delta;acidHi), as shown in Figure 1b. In this equation, &Delta;acidHavg is the average gas-phase acidity of the reference acids, &Delta;(&Delta;S) is the entropy term (which can be assumed constant if the reference acids are structurally similar to each other), R is the universal gas constant, and Teff is the effective temperature of the system. The effective temperature is an empirical parameter that depends on several experimental variables, including the collision energy.
The value of the gas-phase acidity is determined by constructing two sets of thermo-kinetic plots. The first set is obtained by plotting ln([A&macr;]/[Ai&macr;]) against &Delta;acidHi - &Delta;acidHavg, as shown in Figure 4a. Linear regression will yield a set of straight lines with the slopes of X = 1/RTeff and intercepts of Y = - [&Delta;acidH - &Delta;acidHavg]/RTeff - &Delta;(&Delta;S)/R. The second set of plots is obtained by plotting the resulting intercepts (Y) from the first set against the corresponding slopes (X), as shown in Figure 4b. Linear regression produces a new line with a slope of &Delta;acidH - &Delta;acidHavg and an intercept of &Delta;(&Delta;S)/R. The value of &Delta;acidH is then obtained from the slope and the entropy term, &Delta;(&Delta;S), is obtained from the intercept.
The experiments are performed using a triple quadrupole mass spectrometer interfaced to an electrospray ionization (ESI) ion source. A schematic diagram of the mass spectrometer is shown in Figure 2. The CID experiments are performed by mass selecting the proton-bound cluster anions with the first quadrupole unit and allowing them to undergo collisions with argon atoms leaked into the collision chamber which is held at a pressure of around 0.5 mTorr. The dissociation product ions are mass analyzed with the third quadrupole unit. The CID spectra are recorded at several collision energies with the m/z range wide enough to cover all possible secondary fragments. The CID product ion intensities are measured by setting the instrument in the selected reaction monitoring (SRM) mode in which the scan is focused on selected product ions. The CID experiments are performed at four different collision energies, corresponding to center-of-mass energies (Ecm) of 1.0, 1.5, 2.0, and 2.5 eV, respectively. The center-of-mass energy is calculated using the equation: Ecm = Elab [m/(M+m)], where Elab is the collision energy in the laboratory frame, m is the mass of argon, and M is the mass of the proton-bound cluster ion.
In this article, we use the oligopeptide Ala3CysNH2 (A3CH) as the model compound. The C-terminus is amidated and the thiol group (SH) of the cysteine residue will be the acidic site. The selection of the suitable reference acids is crucial for the successful measurement of the gas-phase acidity. The ideal reference acids are structurally similar (to each other) organic compounds with well-established gas-phase acidity values. The reference acids should have acidity values close to that of the peptides. For the peptide A3CH, six halogenated carboxylic acids are chosen as the reference acids. The six reference acids are chloroacetic acid (MCAH), bromoacetic acid (MBAH), difluoroacetic acid (DFAH), dichloroacetic acid (DCAH), dibromoacetic acid (DBAH), and trifluoroacetic (TFAH). Two of them, DFAH and MBAH, will be used to illustrate the protocol.

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			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Mass Spectrometer</td>
			<td>Varian</td>
			<td>1200 L and 320 L</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>402923</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>B56307</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Difluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>142859</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D54702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>242357</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of the gas-phase acidities of oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

<ol>
 <li>The CID bracketing experiments provide information on the relative acidities of the peptide compared to the selected reference acids. Two representative CID spectra of the peptide (A3CH) with two reference acids, DFAH and MBAH, are shown in Figure 3. In Figure 3a the ion abundance (peak height) of the peptide ion is weaker than that of DFA&macr;, and in Figure 3b, the ion abundance of the peptide ion is stronger than that of MBA&macr;. The two spectra suggest that the gas-p...

Watch this Scientific Journal Video about Determination of the Gas-phase Acidities of Oligopeptides at JoVE.com

Determination of the Gas-phase Acidities of Oligopeptides

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

determination-of-the-gas-phase-acidities-of-oligopeptides

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11.0K Views.  University of the Pacific. The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Video: Determination of the Gas-phase Acidities of Oligopeptides

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

From Constructs to Crystals &amp;#8211; Towards Structure Determination of &amp;#946;-barrel Outer Membrane Proteins

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline earth-liquid metal alloys as functions of both composition and temperature in order to acquire thermodynamic data. The cell consists of a chemically stable solid-state CaF2-AF2 electrolyte (where A is the alkaline-earth element such as Ca, Sr, or Ba), with binary A-B alloy (where B is the liquid metal such as Bi or Sb) working electrodes, and a pure A metal reference electrode. Emf data are collected over a temperature range of 723 K to 1,123 K in 25 K increments for multiple alloy compositions per experiment and the results are analyzed to yield activity values, phase transition temperatures, and partial molar entropies/enthalpies for each composition.

This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range.

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they are utilized in a wide variety of applications. In most studies employing photoswitches, several important photophysical properties such as maximum wavelengths of absorption and emission, molar attenuation coefficient, fluorescence lifetime, and photoisomerization quantum yield are carefully determined to investigate their electronic states and transition processes. However, measurement of the photoisomerization quantum yield, the efficiency of photoisomerization with respect to the absorbed photons, in a typical laboratory setting is often complicated and prone to error because it requires the implementation of rigorous spectroscopic measurements and calculations based on an appropriate integration method. This article introduces a set of procedures to measure the photoisomerization quantum yield of a bistable photoswitch using a photochromic hydrazone. We anticipate that this article will be a useful guide for the investigation of bistable photoswitches that are being increasingly developed.

Photoisomerization quantum yield is a fundamental photophysical property that should be accurately determined in the investigation of newly developed photoswitches. Here, we describe a set of procedures to measure the photoisomerization quantum yield of a photochromic hydrazone as a model bistable photoswitch.

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they ...

Analyzing Pesticides in Avocado Using the QuEChERS Method and CG-MS/MS

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of pesticide residues in food. Nevertheless, these methods are vulnerable to matrix effects (MEs), which can potentially affect accurate quantification depending on the specific combination of analyte and matrix. Among the various strategies to mitigate MEs, matrix-matched calibration represents the prevailing approach in pesticide residue applications due to its cost-effectiveness and straightforward implementation. In this study, a total of 45 representative pesticides were analyzed in three different varieties of avocado (i.e., Criollo, Hass, and Lorena) using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate and GC-MS/MS. 
For this purpose, 5 g of the avocado sample was extracted with 10 mL of acetonitrile, and then 2.5 g of ammonium formate was added to induce phase separation. Subsequently, the supernatant underwent a cleanup process via dispersive solid-phase extraction employing 150 mg of anhydrous MgSO4, 50 mg of primary-secondary amine, 50 mg of octadecylsilane, 10 mg of graphitized carbon black, and 60 mg of a zirconium oxide-based sorbent (Z-Sep+). The GC-MS/MS analysis was successfully performed in less than 25 min. Rigorous validation experiments were carried out to assess the performance of the method. The examination of a matrix-matched calibration curve for each variety of avocado revealed that the ME remained relatively consistent and less than 20% (considered as a soft ME) for most pesticide/variety combinations. Furthermore, the method&#180;s limits of quantification were lower than 5 &#181;g/kg for all three varieties. Finally, the recovery values for most pesticides fell within the acceptable range of 70-120%, with relative standard deviation values below 20%.

Avocado Sample Preparation Using the QuEChERS Method with Ammonium Formate for Pesticide Analysis

The present protocol describes the analysis of multiclass pesticide residues in avocado varieties using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate, followed by gas chromatography-tandem mass spectrometry.