This work has been performed at the Molecular Foundry, Lawrence Berkeley National Laboratory and supported by the Office of Science, Office of Basic Energy Sciences, Scientific User Facilities Division of the US Department of Energy, under Contract No. DE-AC02&#8211;05CH11231. The financial support of F.M. by a ME-Fulbright fellowship and A.S. by Higher Education Commission of Pakistan are gratefully acknowledged.

NOTE: Before beginning, check all relevant material data sheets (MSDSs). Several of the chemicals used in the synthetic and application procedures are toxic. Please follow all appropriate safety practices and use adequate protective equipment (lab coat, full-length pants, closed-toe shoes, safety glasses, gloves). Please use all cryogenic personal protective equipment when handling liquid nitrogen for the nitrogen adsorption measurements (insulated gloves, face shield).
1. Porous Polymer Monolith Preparation in Bulk and Capillary Column Format
<ol>
	<li>Bulk Polymer Monolith for Characterization
	<ol>
		<li>Purify styrene, divinylbenzene and methacrylic acid through a column of basic alumina, in order to remove the polymerization inhibitors. Place 10 g of basic alumina in a 25 ml disposable plastic syringe with a plug of glass wool fiber packed in the syringe tip. Percolate approximately 10 ml of the monomer through the column.</li>
		<li>Load the monomers (50 mg styrene, 100 mg divinylbenzene and 50 mg methacrylic acid) and the pore forming agents (300 mg toluene and 300 mg isooctane) in a 1 ml glass vial. Add the initiator of the polymerization, 4 mg of 2,2&#8217;-azobisisobutyronitrile (AIBN, 1% with respect to monomers).</li>
		<li>Homogenize by sonication for 10 min. Remove dissolved oxygen by bubbling nitrogen through the liquid for 10 min. Seal the vial cap with paraffin film and place it in a water bath at 60&#160;&#176;C for 6 hr to polymerize the mixture.</li>
		<li>Cool to room temperature and break the vial carefully. Transfer the polymer monolith into a cellulose extraction thimble. Place the extraction thimble into a Soxhlet extraction chamber and assemble it to a round bottom flask that contains a volume of methanol, which is at least three times the volume of the extraction chamber. Assemble a condenser to the upper part of the extraction chamber. Perform Soxhlet extraction by boiling the methanol for 16 hr, ensuring the complete removal of the unreacted monomers and pore forming agents.</li>
		<li>Dry overnight in a vacuum oven at 60 &#176;C. Confirm the presence of carboxylic functional groups to attach the PCP by Fourier Transform Infrared Spectroscopy (FT-IR). Measure surface area by nitrogen adsorption porosimetry.</li>
	</ol>
	</li>
	<li>Functionalization of Silica Capillaries for the Preparation of Monolithic Columns
	<ol>
		<li>Cut 2 m of a polyimide-coated 100 &#181;m i.d. fused silica capillary. Connect it to a 0.25-0.50 ml glass syringe and wash the capillary with acetone. Remove the acetone by rinsing the capillary with water.</li>
		<li>In order to activate the internal silica coating of the capillary, use a syringe pump to flow a 0.2 M aqueous NaOH solution at 0.25 &#181;l/min for 30 min. Rinse with water until the effluent is neutral.</li>
		<li>Use pH paper strips to check effluent pH. In order to protonate the silanol groups of the capillary, pump a 0.2 M aqueous HCl solution through the capillary at 0.25 &#181;l/min for 30 min. Rinse with water until the effluent is neutral. Rinse with ethanol.</li>
		<li>Pump a 20% (w/w) ethanol solution of 3-(trimethoxysilyl)propyl methacrylate (pH 5 adjusted with acetic acid) at 0.25 &#181;l/min for 1 hr. In this step, the silica capillary is functionalized with vinyl groups in order to attach the polymer monolith to the capillary inner surface.</li>
		<li>Rinse with acetone, dry in a nitrogen stream and leave at room temperature overnight before use. Cut the capillary into shorter pieces of length 20 cm.</li>
	</ol>
	</li>
	<li>Preparation of Monolithic Capillary Columns
	<ol>
		<li>Prepare an identical polymerization mixture as for the bulk polymer monolith (section 1.1) in a 1 ml glass vial with a rubber septum. Add initiator 1% AIBN with respect to monomers. Homogenize by sonication for 10 min.</li>
		<li>Purge the polymerization mixture with nitrogen by coupling a non-functionalized silica capillary to a nitrogen stream.
		<ol>
			<li>Insert the nitrogen stream capillary through the rubber septum of the vial and immerse it into the polymerization mixture so that the nitrogen bubbles through the liquid. Leave the vial cap slightly loose to avoid overpressure. Purge for 10 min.</li>
			<li>Lift the nitrogen stream capillary from the polymerization mixture to the headspace of the vial, and close tightly the cap. Insert a functionalized capillary through the septum into the polymerization mixture. The excess of pressure generated into the capillary through the nitrogen injected into the headspace pumps the polymerization mixture through the functionalized capillary.</li>
			<li>Collect several drops of polymerization mixture from the effluent of the capillary to ensure that it is completely filled and close it with a rubber septum. Take the capillary out of the vial very carefully and close the inlet of the capillary with a rubber septum.</li>
		</ol>
		</li>
		<li>Polymerize the mixture contained in the capillary in a water bath at 60 &#176;C for 6 hr. Cool at room temperature and cut a few millimeters of both ends of the capillary. Remove unreacted monomers and pore forming agents by flushing the column with acetonitrile using an HPLC pump at 3 &#181;l/min for 30 min. Check backpressure of the capillary column.</li>
	</ol>
	</li>
</ol>
2. Growth of the Iron-benzenetrycarboxylate (FeBTC) PCP
<ol>
	<li>Growth of the FeBTC MOPH on a Bulk Polymer Monolith for Characterization
	<ol>
		<li>Grind the previously dried monolith using a mortar and pestle.</li>
		<li>Immerse 100 mg of the monolith powder in 5 ml of 2 mM FeCl3&#183;6H2O in ethanol for 15 min. Vacuum filter using a nylon filter (0.22 &#181;m) and wash the powder with ethanol. Immerse the monolith powder in 5 ml of 2 mM 1,3,5-benzenetricarboxylic acid (BTC) in ethanol for 15 min. Vacuum filter using a nylon filter (0.22 &#181;m) and wash the powder with ethanol.</li>
		<li>Repeat step number 2 as desired. The growth of the final metal-organic coating will be defined by the number of applied cycles. Typically, between 10 and 30 cycles are performed. Confirm the presence of new pores by nitrogen adsorption porosimetry. Measure the amount of additional metal sites by thermogravimetric analysis (TGA).</li>
	</ol>
	</li>
	<li>Growth of the FeBTC MOPH on a capillary monolithic column for the enrichment of phosphopeptides
	<ol>
		<li>Using a syringe pump. Flush the capillary monolith with 2 mM FeCl3&#183;6H2O in ethanol for 15 min at 2 &#181;l/min. Wash with ethanol for 15 min at 2 &#181;l/min. Flush the capillary monolith with a 2 mM BTC in ethanol for 15 min at 2 &#181;l/min. Wash with ethanol for 15 min at 2 &#181;l/min.</li>
		<li>Repeat step 1 as desired. The growth of the final metal-organic coating will be defined by the number of cycles performed.</li>
	</ol>
	</li>
</ol>
3. Protein Digestion and Enrichment of Phosphopeptides
<ol>
	<li>Protein Digestion
	<ol>
		<li>Dissolve 0.5 ml of non-fat milk in 1 ml of water and divide it into 200 &#181;l fractions.</li>
		<li>For the protein digestion add 160 &#181;l 1 M ammonium bicarbonate and 50 &#181;l 45 mM dithiothreitol to each fraction, in order to cleave the disulfide bonds. Incubate at 50 &#176;C in a thermomixer for 15 min.</li>
		<li>Add gradually 50 &#181;l of an aqueous solution of iodoacetamide 100 mM, while the solution cooled down to room temperature. Iodoacetamide will prevent the formation of new disulfide bonds.</li>
		<li>Incubate in the dark for 15 min at room temperature. Add 1 ml deionized water. Add 2 &#181;g trypsin and digest proteins in a thermomixer at 37 &#176;C for 14 hr.</li>
		<li>Terminate digestion by acidification with 10 &#181;l of 1% trifluoroacetic acid, and placing it in the thermomixer for 5 min at room temperature. Store the digested proteins at &#8211;20 &#176;C.</li>
	</ol>
	</li>
	<li>Enrichment of phosphopeptides using a capillary MOPH column.
	<ol>
		<li>Flush the column with 100 &#181;l of a 4:1 mixture of acetonitrile containing a 0.1% trifluoroacetic acid for 10 min at a flow rate of 1 &#181;l/min. Pump the protein digestion through the column at 2 &#181;l/min for 30 min.</li>
		<li>Wash out the non-phosphorylated peptides again with a 4:1 mixture of acetonitrile containing a 0.1% trifluoroacetic acid for 10 min at a flow rate of 1 &#181;l/min. Wash with water for 10 min at a flow rate of 1 &#181;l/min.</li>
		<li>Elute phosphopeptides using a 250 mM pH 7 phosphate buffer solution pumped at 1 &#181;l/min for 15 min. Collect the eluent in a vial and desalt the solution using a standard protocol19. Prepare a 2 mg/ml 2,5-dihydroxybenzoic acid to use it as the matrix for the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Drawn 2 &#181;l of the 2,5-dihydroxybenzoic acid into the tip to elute the phosphopeptides and spot them directly on to the MALDI plate.</li>
		<li>Analyze the spots by MALDI-TOF-MS and regenerate the column by flushing thoroughly with water and then methanol.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The original polymer monolith contains carboxylic functional groups able to bind to metals. Coordinating the initial metal sites on the original material, we are able to grow a PCP coating (Figure 1A), incorporating a number of additional metal sites shaping a microporous network. This makes the presented MOPH materials attractive for extraction or purification procedures where metallic species are involved, such as the immobilized metal-ion affinity chromatography (IMAC) technique. The general procedure...

Porous coordination polymers (PCPs) are coordination compounds based on metal centers linked by organic ligands with repeating coordination entities extending in 1, 2 or 3 dimensions that can be amorphous or crystalline1-3. In recent years, this class of porous materials has attracted widespread attention due to their high porosity, wide chemical tunability, and their stability. PCPs have been explored for a range of applications including gas storage, gas separation, and catalysis3-6, and very recently, the first analytical applications of PCPs have been described7.
Because of their enhanced chemical functionality and high porosity PCPs have been targeted for their huge potential for the improvement of purification processes and chromatographic separations, and a number of reports concerning this topic have been published7-13. However, the performance of PCPs is not currently at an equivalent level with existing chromatographic materials likely due to fast diffusion through large interparticle voids in packed beds of these solids due to their typically irregularly shaped morphologies of their particles or crystals. This irregularly distributed packing leads to a lower than expected performance, as well as high column backpressures and undesirable peak shape morphologies14,15.
In order to solve the problem of fast diffusion through the inter-particle voids and concomitantly enhance the performance of PCPs for analytical applications, the development of a hybrid material based on a macroporous polymer monolith16 that contains the PCP on the surface of the macropores would be desirable. Polymer monoliths are self-contained, single-piece materials that can sustain convective flow through their pores, which makes them one of the most efficient alternatives to bead packings and have been successfully commercialized by several companies17,18.&#160;Porous polymer monoliths are usually based on the polymerization of a monomer and a crosslinker in the presence of porogens, which are typically binary mixtures of organic solvents. The obtained monolithic materials have a microglobular structure and a high porosity and flow permeability.
A simple approach to unify these materials to prepare a polymer monolith containing a PCP is based on the direct addition of as-synthesized PCPs in the polymerization mixture of the monolith. This approach resulted in PCPs mostly buried within a polymer scaffold, and not being active for the further application of the final material14,15. A different synthetic approach is clearly needed in order to, for example, develop uniform films of PCPs, or crystalline metal-organic frameworks (MOFs) where the majority of the pores contained within the crystal are accessible from the macropores of the polymer monolith.
Herein we report a simple protocol for the preparation of a metal-organic polymer hybrid material (MOPH) based on a macroporous polymer support with suitable functional groups for the attachment of PCPs, which can be easily implemented as a self-contained single-piece polymer monolith in a column format with optimum properties for flow-through applications. The polymer synthesis procedure is followed by a simple room temperature solution-based&#160;method to grow a PCP coating on the internal surface of the pores of the monolith19-20. As the first example, we describe the preparation of an iron(III) benzenetricarboxylate (FeBTC) coordination polymer film within a macroporous poly(styrene-divinylbenzene-methacrylic acid) monolith. This method is effective for the preparation of bulk powders as well as capillary columns and the described protocol is readily implementable to other PCPs. As an example of the potential of MOPHs as functional materials for flow-through applications, we applied the developed FeBTC MOPH which contains a dense coating of Fe(III) centers to enrich phosphopeptides from digested protein mixtures exploiting the binding affinity of phosphopeptides to Fe(III). The developed protocol21 comprises three main parts: Preparation of the macroporous organic polymer monolith support; growth of the PCP coating on the surface of the pores of the monolith; application for the enrichment of phosphopeptides.

<table><tbody><tr><td>Polyimide-coated capillaries</td><td>Polymicro Technologies</td><td>TSP100375</td><td>100 &amp;mu;m i.d.</td></tr><tr><td>3-(trimethoxysilyl)propyl methacrylate, 98%</td><td>Sigma-Aldrich</td><td>440159</td><td></td></tr><tr><td>Styrene, 99%</td><td>Sigma-Aldrich</td><td>W323306</td><td>Technical grade</td></tr><tr><td>Divinylbenzene, 80%</td><td>Sigma-Aldrich</td><td>414565</td><td></td></tr><tr><td>Methacrylic acid, 98%</td><td>Mallinckrodt</td><td>MK150659</td><td></td></tr><tr><td>Toluene, &amp;ge;99.5%</td><td>EMD chemicals</td><td>MTX0735-6</td><td></td></tr><tr><td>Isooctane, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>650439</td><td></td></tr><tr><td>2,2&#039;-azobisisobutyronitrile, 98%</td><td>Sigma-Aldrich</td><td>441090</td><td></td></tr><tr><td>Aluminium oxide (basic alumina)</td><td>Sigma-Aldrich</td><td>199443</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate, 97%</td><td>Sigma-Aldrich</td><td>236489</td><td></td></tr><tr><td>1,3,5-benzenetrycarboxylic acid, 95%</td><td>Sigma-Aldrich</td><td>482749</td><td></td></tr><tr><td>Acetonitrile, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>360457</td><td></td></tr><tr><td>Ammonium bicarbonate, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr><tr><td>Trifluoroacetic acid, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>302031</td><td></td></tr><tr><td>Ethanol, &amp;ge;99.8%</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Iodoacetamide, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>Dithiothreitol, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>Monobasic sodium phosphate dihydrate, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>71505</td><td></td></tr><tr><td>Dibasic sodium phosphate dihydrate, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>Phosphoric acid, &amp;ge;85%</td><td>Sigma-Aldrich</td><td>438081</td><td></td></tr><tr><td>2,5-dihydroxybenzoic acid, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>85707</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T8003</td><td>Bovine pancreas</td></tr><tr><td>&amp;beta;-casein</td><td>Sigma-Aldrich</td><td>C6905</td><td>Bovine milk</td></tr><tr><td>ZipTip pipette tips</td><td>Merck Millipore</td><td>ZTC18S096</td><td>C18 resin</td></tr></tbody></table>

preparation of highly porous coordination polymer coatings on macroporous polymer monoliths for enhanced enrichment of phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

A schematic illustration of the PCP growth on the pore surface of the organic polymer monolith is shown in Figure 1. In this figure, we illustrate the initial Fe(III) atoms retained on the pore surface of the original polymer monolith coordinated to carboxylic functional groups. Using the protocol described herein additional organic ligand and Fe(III) ions are added to the surface, shaping a porous coordination network within the polymer monolith. Figure 1 also shows schematically the us...

Watch this Scientific Journal Video about Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides at JoVE.com

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

preparation-highly-porous-coordination-polymer-coatings-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

10.0K Views.  E. O. Lawrence Berkeley National Laboratory. A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

Video: Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.

We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days.

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma ...

Engineering

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Cancer Research

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

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 ...

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Bioengineering

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...