The authors thank Prof. Thomas Reiner, Dr. Jacob Houghton, and Dr. Serge Lyaschenko for helpful conversations.&#160;

All of the in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee (IACUC).
1. Conjugation of DFO-NCS to J591
<ol>
	<li>In a 1.7 ml microcentrifuge tube, prepare a 2-5 mg/ml solution of J591 in 1 ml of either 1x phosphate-buffered saline (pH 7.4) or 0.5 M HEPES buffer (pH 7.4).</li>
	<li>Dissolve DFO-NCS in dry DMSO at a concentration between 5-10 mM (3.8-7.6 mg/ml). Sonicate or vortex the solution thoroughly in order to facilitate complete dissolution.</li>
	<li>Adjust the pH of the J591 solution to 8.8-9.0 by adding small aliquots (&#60; 10 &#181;l) of 0.1 M Na2CO3.</li>
	<li>Once the antibody solution is at the correct pH, add a volume of the DFO-NCS solution corresponding to a 3-4-fold molar excess of the bifunctional chelator.
	<ol>
		<li>For example, add 4-5 &#181;l of a 10 mM (7.6 mg/ml) DFO-NCS solution (40.4 nmol DFO-NCS) to 1 ml of a 2 mg/ml J591 antibody solution (13.3 nmol J591). The amount of DMSO in the final aqueous reaction mixture should not exceed 2% v/v.</li>
	</ol>
	</li>
	<li>Incubate the reaction for 30 min at 37 &#176;C on an agitating heating block at 350 rpm.</li>
	<li>After 1 hr at 37 &#176;C, purify the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50,000 molecular weight cut-off using 0.5 M HEPES buffer (pH 7.4) as the eluent. This step will yield a 2 ml solution of the completed J591-DFO construct.</li>
	<li>Measure the concentration of the J591-DFO construct on a UV-Vis spectrophotometer.</li>
	<li>If a higher concentration of the construct is desired, concentrate the J591-DFO solution using a centrifugal filter unit with a 50,000 molecular weight cut-off.</li>
	<li>Store the solution of the completed J591-DFO immunoconjugate at -20 &#176;C in the dark. 
	NOTE: This is an acceptable stopping point in the procedure.</li>
</ol>
2. Radiolabeling J591-DFO with 89Zr
CAUTION: This step of the protocol involves the handling and manipulation of radioactivity. Before performing these steps or performing any other work with radioactivity researchers should consult with their home institution&#8217;s Radiation Safety Department. All possible steps should be taken to minimize exposure to ionizing radiation. &#160;
NOTE: In the interest of proper radiochemical note-keeping, the amount of radioactivity in the sample should be measured using a dose calibrator and recorded before and after Steps 2.2-2.13 in the protocol below. This will help with the accurate determination of radiochemical yields and specific activities.
<ol>
	<li>Prepare a solution of 0.5-2.0 mg of J591-DFO in 200 &#181;l of 0.5 M HEPES buffer, pH 7.5.</li>
	<li>Pipette a volume of the 89Zr4+ stock solution (typically supplied in 1.0 M oxalic acid) corresponding to 1.0-6.0 mCi (37-222 MBq) into a 2 ml plastic screw-cap microcentrifuge tube. Adjust the volume of this solution to a total 300 &#181;l using 1.0 M oxalic acid.</li>
	<li>Adjust the pH of the 89Zr4+ solution to 6.8-7.5 using 1.0 M Na2CO3. Begin by adding 250 &#181;l of 1.0 M Na2CO3 to the 89Zr4+ solution and subsequently add smaller (&#60; 10 &#181;l) aliquots of base to achieve the desired pH.</li>
	<li>Add the desired amount of pH-adjusted 89Zr4+ solution to the J591-DFO solution prepared in Step 2.1.</li>
	<li>Check the pH of the radiolabeling reaction mixture to ensure that it falls within the desired range of 6.8-7.5.</li>
	<li>Incubate the radiolabeling reaction for 60 min at RT on an agitating heating block at 350 rpm.</li>
	<li>After 60 min of incubation, measure the radiolabeling yield of the reaction using radio-TLC.
	<ol>
		<li>To this end, spot 1 &#181;Ci of the radiolabeling reaction mixture on a silica-impregnated TLC strip. Allow the aliquot to dry, run the TLC using an eluent of 50 mM DTPA (pH 5.5) and analyze the TLC strip using a radio-TLC scanner. 89Zr4+ bound to the J591-DFO construct will appear at the origin (Rf &#60; 0.1), while free 89Zr4+ cations will be chelated by DTPA and will elute with the solvent front (Rf &#62; 0.9).</li>
		<li>Calculate the radiolabeling yield of the reaction by integrating the radiochromatogram, dividing the area under the curve from Rf 0.0-0.1 by the total area under the curve, and multiplying by 100.</li>
	</ol>
	</li>
	<li>If the radiolabeling yield is sufficient (typically a theoretical specific activity of &#62;2 mCi/mg), quench the reaction with 5 &#181;l of 50 mM DTPA, pH 5.5.</li>
	<li>Purify the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50,000 molecular weight cut-off using an eluent of either 0.9% sterile saline with 5 mg/ml gentisic acid or 0.25 M sodium acetate (pH 5.5) with 5 mg/ml gentisic acid. This step will yield a 2 ml solution of the completed 89Zr-DFO-J591 radioimmunoconjugate.</li>
	<li>After purification, verify the radiochemical purity of the 89Zr-DFO-J591 using radio-TLC as described in Step 2.7.</li>
	<li>Calculate the overall radiolabeling yield of the reaction by dividing the amount of activity initially added to the antibody solution by the amount of radioactivity isolated with the purified 89Zr-DFO-J591 radioimmunoconjugate.</li>
	<li>Calculate the final specific activity by dividing the amount of activity isolated with the purified 89Zr-DFO-J591 radioimmunoconjugate by the initial mass of DFO-J591 in the radiolabeling reaction.</li>
	<li>If a higher concentration is desired, concentrate the 89Zr-DFO-J591 solution using a centrifugal filter unit with a 50,000 molecular weight cut-off. 
	NOTE: The gentisic acid used in the final purification step is a radio-protectant employed to minimize the degradation of the antibody due to radiolysis.46 While the storage of the 89Zr-DFO-J591 radioimmunoconjugate for up to 48 hr at 4 &#176;C is possible, it is not recommended. If the radioimmunoconjugate is to be stored, use 0.25 M sodium acetate (pH 5.5) with 5 mg/ml gentisic acid as the storage buffer in order to minimize the risk of hypochlorite-mediated radiolysis.47</li>
</ol>
3. In Vivo PET Imaging with 89Zr-DFO-J591
CAUTION: As in Protocol Section 2, this step of the protocol involves the handling and manipulation of radioactivity. Before performing these steps researchers should consult with their home institution&#8217;s Radiation Safety Department. All possible steps should be taken to minimize exposure to ionizing radiation.
<ol>
	<li>In male athymic nude mice, subcutaneously implant 5 x 106 LNCaP prostate cancer cells and allow these to grow to a 100-150 mm3 xenograft (3-4 weeks after inoculation).44</li>
	<li>Dilute the 89Zr-DFO-J591 radioimmunoconjugate to a concentration of 1.0 mCi/ml in 0.9% sterile saline.</li>
	<li>Inject 200 &#181;l of the 89Zr-DFO-J591 solution (200 &#181;Ci; 7.4 MBq) into the lateral tail vein of the xenograft-bearing mice.48</li>
	<li>At the desired imaging time point (e.g., 12, 24, 48, 72, 96, or 120 hr post-injection), anesthetize the mouse with a 2% isoflurane: oxygen gas mixture.</li>
	<li>Place the mouse on the bed of the small animal PET scanner, and maintain anesthesia during the scan using a 1% isoflurane: oxygen gas mixture. Prior to placing the animal on the scanner bed, verify anesthesia using the toe-pinch method and apply ophthalmic&#160;ointment to the eyes of the mouse to prevent drying during anesthesia.49</li>
	<li>Acquire the PET data for the mouse via a static scan with a minimum of 40 million coincident events using an energy window of 350-700 keV and a coincidence timing window of 6 nsec.50</li>
	<li>After completing the acquisition of the image, do not leave the mouse unattended and do not place it in a cage with other mice until it has regained consciousness.</li>
</ol>

The authors have nothing to disclose.

While the construction, radiolabeling, and imaging of 89Zr-DFO-labled radioimmunoconjugates is generally a rather straightforward procedure, it is important to keep a few key considerations in mind during each step of the process. For example, perhaps the most likely cause for concern during the conjugation step of the procedure is the aggregation of the antibody during the conjugation reaction. This problem is most often a product of poor mixing of the conjugation reaction after the addition of the DFO-NCS st...

Due to their remarkable sensitivity, affinity, and selectivity, antibodies have long been considered promising vectors for the delivery of radioisotopes to cancer cells. However, their application in positron emission tomography (PET) imaging has been hampered by the lack of a suitable positron-emitting radioisotope for their labeling.1-3 One of the most critical considerations in the design of radioimmunoconjugates is matching the physical decay half-life of the radioisotope to the in vivo pharmacokinetics of the antibody. More specifically, antibodies often have relatively long, multi-day biological half-lives and therefore must be labeled with radioisotopes with comparable physical half-lives. For PET imaging applications, antibodies have traditionally been radiolabeled with 64Cu (t1/2 = 12.7 hr), 86Y (t1/2 = 14.7 hr), or 124I (t1/2 = 4.18 d).4,5 However, each of these radioisotopes possesses significant limitations that hamper their suitability for clinical imaging. While radioimmunoconjugates labeled with 86Y and 64Cu have proven promising in preclinical investigations, both isotopes possess physical half-lives that are too short to be effective for imaging in humans. 124I, in contrast, has a nearly ideal physical half-life for imaging with antibodies, but it is expensive and has suboptimal decay characteristics that lead to relatively low resolution clinical images. Furthermore, 124I-labeled radioimmunoconjugates can be subject to dehalogenation in vivo, a process which can lower tumor-to-background activity ratios.6,7
The drive to find a positron-emitting radioisotope to supplant 64Cu, 86Y, and 124I in radioimmunoconjugates has fueled the recent surge in research on 89Zr-labeled antibodies.8-12 The reason for the advent of 89Zr is straightforward: the radiometal possesses near-ideal chemical and physical properties for use in diagnostic PET radioimmunoconjugates.13 89Zr is produced via the 89Y(p,n)89Zr reaction on a cyclotron using a commercially available and 100% naturally abundant 89Y target.14,15 The radiometal has a positron yield of 23%, decays with a half-life of 78.4 hr, and emits positrons with the relatively low energy of 395.5 keV (Figure 1).13,16,17 It is important to note that 89Zr also emits a high energy, 909 keV &#947;-ray with 99% efficiency. While this emission does not interfere energetically with the emitted 511 keV photons, it does require extra consideration with regard to transport, handling, and dosimetry. Despite this caveat, these decay characteristics ultimately mean that 89Zr not only has a more favorable half-life for imaging with antibodies than 86Y and 64Cu but can also produce higher resolution images than 124I, which emits positrons with higher energies of 687 and 975 keV as well as a number of photons with energies within 100-150 keV of the 511 keV positron-created photons.13 Moreover, 89Zr is also safer to handle, less expensive to produce, and residualizes in tumors more effectively than its radioiodine counterpart.18,19 One potential limitation of 89Zr is that it does not have a therapeutic isotopologue, e.g., 86Y (PET) vs. 90Y (therapy). This precludes the construction of chemically identical, surrogate imaging agents that can be employed as dosimetric scouts for their therapeutic counterparts. That said, investigations suggest that 89Zr-labeled antibodies do have potential as imaging surrogates for 90Y- and 177Lu-labeled immunoconjugates.20,21
From a chemical standpoint, as a Group IV metal, 89Zr exists as a +4 cation in aqueous solution. The Zr4+ ion is highly charged, relatively large (effective ionic radius = 0.84 &#197;), and can be classified as a &#8220;hard&#8221; cation. As such, it exhibits a preference for ligands bearing up to eight hard, anionic oxygen donors. Easily the most common chelator used in 89Zr-labeled radioimmunoconjugates is desferrioxamine (DFO), a siderophore-derived, acyclic chelator bearing three hydroxamate groups. The ligand stably coordinates the Zr4+ cation quickly and cleanly at RT at biologically relevant pH levels, and the resulting Zr-DFO complex remains stable over the course of multiple days in saline, blood serum, and whole blood.22 Computational studies strongly suggest that DFO forms a hexacoordinate complex with Zr4+ in which the metal center is coordinated to the three neutral and three anionic oxygen donors of the ligand as well as two exogenous water ligands (Figure 2).23,24 The in vivo behavior of radioimmunoconjugates employing the 89Zr-DFO conjugation scaffold has generally been excellent. However, in some cases, imaging and acute biodistribution studies have revealed elevated activity levels in the bones of mice injected with 89Zr-labeled antibodies, data that suggests that the osteophilic 89Zr4+ cation is released from the chelator in vivo and subsequently mineralizes in the bone.25 Recently, a number of investigations into the development of novel 89Zr4+ chelators particularly ligands with eight oxygen donors have appeared in the literature.24,26,27 Nevertheless, at present, DFO is the most widely employed chelator in 89Zr-labeled radioimmunoconjugates by a wide margin. A variety of different bioconjugation strategies have been employed to attach DFO to antibodies, including bioorthogonal click chemistry, the reaction of thiol-reactive DFO constructs with cysteines in the antibody, and the reaction of activated ester-bearing DFO constructs with lysines in the antibody.4,28-30 Easily the most common strategy, however, has been the use of an isothiocyanate-bearing derivative of DFO, DFO-NCS (Figure 2).22 This commercially available bifunctional chelator robustly and reliably forms stable, covalent thiourea linkages with the lysines of the antibody (Figure 3).
Over the past few years, a wide variety of 89Zr-DFO-labeled radioimmunoconjugates have been reported in the literature. Preclinical investigations have been especially abundant, featuring antibodies ranging from the more well-known cetuximab, bevacizumab, and trastuzumab to more esoteric antibodies such as the CD105-targeting TRC105 and fPSA-targeting 5A10.30-36 More recently, a small number of early-phase clinical trials using 89Zr-DFO-labeled antibodies have emerged in the literature. Specifically, groups in the Netherlands have published trials employing 89Zr-DFO-cmAb U36, 89Zr-DFO-ibritumomab tiuxetan, and 89Zr-DFO-trastuzumab.21,32,37 In addition, a range of other clinical trials with 89Zr-labeled radioimmunoconjugates are currently underway, including investigations here at Memorial Sloan Kettering Cancer Center using the PSMA-targeting 89Zr-DFO-J591 for prostate cancer imaging and the HER2-targeting 89Zr-DFO-trastuzumab for breast cancer imaging.23,30 In addition, while radiolabeled antibodies remain the most common 89Zr-labeled radiopharmaceuticals, the radiometal has also increasingly been employed with other vectors, including peptides, proteins, and nanomaterials.38-43
The modularity of this 89Zr-DFO labeling methodology is a tremendous asset. The repertoire of biomarker-targeting antibodies is ever-expanding, and the interest in performing in vivo PET imaging using these constructs is growing apace. As a result, we believe that the development of more standardized practices and protocols could benefit the field. An excellent written experimental protocol for DFO-NCS conjugation and 89Zr radiolabeling has already been published by Vosjan, et al.22 We feel that the visual demonstration provided by this work could further help investigators new to these techniques. In the protocol at hand, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of the 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with a 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.23,44,45

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Name of the Material/Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td style="width:179px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-SCN-Bn-DFO</td>
			<td>Macrocyclics</td>
			<td>B-705</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">[89Zr]Zr-oxalate</td>
			<td>Various, including Perkin-Elmer</td>
			<td>-</td>
			<td>Caution: Radioactive material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>17-0851-01&#160;</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td>EMD Millipore</td>
			<td>UFC805024</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica Gel Impregnanted RadioTLC Paper</td>
			<td>Agilent Technologies</td>
			<td>SGI0001</td>
			<td>Cut into strips 0.5 cm wide</td>
		</tr>
	</tbody>
</table>

the bioconjugation and radiosynthesis of 89zr-dfo-labeled antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The first step in this protocol the conjugation of DFO-NCS to the antibody is typically quite robust and reliable. Generally, the purified, chelator-modified immunoconjugate can be obtained in &#62; 90% yield, and using 3 molar equivalents of DFO-NCS in the initial conjugation reaction will yield a degree-of-labeling of the chelator of approximately 1.0-1.5 DFO/mAb. The 89Zr radiolabeling and purification steps of the procedure are likewise straightforward. At the concentrations outlined in the protocol above,...

Watch this Scientific Journal Video about The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies at JoVE.com

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

the-bioconjugation-and-radiosynthesis-of-89zr-dfo-labeled-antibodies

Microsoft Bing

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Chemistry

Unit 1

Fundamentals of Chemistry

SCIENTISTS IN ACTION

17.0K Views.  Memorial Sloan Kettering Cancer Center. Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described. 

Video: The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

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 ...

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...