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

The overall goal of the following experiment is to prepare a novel support material with improved performance in the selective extraction of phospho peptides. This is achieved by proper preparation of a porous polymer capillary monolith with suitable functional groups. First, a metal species is immobilized on the functional groups of a polymer monolith, making the support selective to the extraction of phospho peptides.

Next step-by-step growth of multiple layers of the metal organic framework is performed in order to increase the amount of metal sites, thus increasing the selectivity for phospho peptides. The results show improved selectivity for the extraction of phospho peptides from protein digests based on matrix assisted laser desorption ionization, time of flight mass spectrometry. The enrichment of O peptide is usually done by metal lion affinity chromatography, which is performed on either conventional silica particles or polymer particles, which are packed into a chromatographic column.

Our material combines the advantages of a macroporous polymer monolith, which has good mass transport properties with the high binding capacity of a metal organic framework. The way we achieve this is by sequentially growing the metal organic framework from the surface of the monolith so that in the end we have multiple layers of metal lines that are available to interact with the analyte Visual demonstration of the polymer monolith preparation and metal organic framework growth are both important because they're very difficult to learn. There are several important steps that one has to pay attention to in order to achieve a monolith with good surface properties and porosity.

To begin, cut two meters of a fused silica capillary that is polyamide coated and has an inner diameter of 100 microns connected to a point 25 milliliter glass syringe and wash the capillary with acetone. Then remove the acetone by rinsing the capillary with water. Next place a 250 microliter glass syringe filled with a 0.2 molar aqueous sodium hydroxide onto the syringe pump and flow the solution at point 25 microliters per minute through the capillary for 30 minutes in order to activate the internal silica coating.

After 30 minutes, rinse the capillary with water until the effluent is neutral. Use paper pH strips to verify the neutral pH flow. Next load to syringe with 0.2 molar aqueous hydrogen chloride and pump the solution through the capillary at point 25 microliters per minute to protonate the ciol groups after 30 minutes of flow, again, rinse the capillary with water until the flow is neutral.

Once the pH is neutral, rinse the capillaries with a 250 microliter syringe filled with 100%ethanol. Then pump a 20%solution of three trimethyl propyl methacrylate in ethanol at point 25 microliters per minute for one hour to functionalize the surface with vinyl groups. Next, rinse the capillaries with acetone and then dry them with a stream of nitrogen.

Let the samples rest at room temperature overnight, and then cut them into 20 centimeter long sections. Prepare the polymerization mixture in a one milliliter glass vial with a rubber septum as described in the accompanying text protocol. Add 1%A IBN with respect to the monomers as an initiator and sonicate the mixture for 10 minutes.

Next, attach a non functionalized silica capillary to a nitrogen stream and insert it through the rubber septum into the polymerization mixture. Loosen the vial cap slightly and slowly bubble nitrogen through the mixture for 10 minutes. After 10 minutes, lift the nitrogen stream capillary from the polymerization mixture to the headspace of the vial and close the cap tightly.

Insert one of the functionalized capillaries through the septum into the polymerization mixture. Use the excess of pressure generated by the nitrogen to pump the polymerization mixture up into the functionalized capillary. Allow several drops of the polymerization mixture to flow from the effluent of the capillary to ensure that it is completely filled, and then close it with a rubber septum.

Take the capillary out of the vial very carefully and close the inlet with a rubber septum placed a mixture contained in the capillary in a heated water bath at 60 degrees Celsius for six hours to polymerize. When finished, cool the capillary to room temperature and then cut a few millimeters off both ends of the capillary. Next, use an HPLC pump to flush the column with Acetonitrile at four microliters per minute for 30 minutes.

This will remove any unreactive, monomers and poor forming agents. Check the back pressure of the capillary column. Next, flush the capillary monolith in cycles with the reagents shown here to grow the final metal organic coating.

The coating thickness is determined by the number of cycles performed. Flush the capillaries with the prepared metal organic coating using 100 microliters of a four to one mixture of acetonide trial and 0.1%aqueous trior acetic acid. Then pump a protein digest through the column at two microliters per minute.

For 30 minutes, wash out the non phosphorylated peptides again with a four to one mixture of acetyl NI trial and 0.1%aqueous Tri Fluor acetic acid for 10 minutes at a flow rate of one microliter per minute. Wash with water for 10 minutes at a flow rate of one microliter per minute. Elute the phospho peptides using a 250 micromolar phosphate buffer solution at pH seven by pumping it through the capillary at one microliter per minute for 15 minutes, collect the ellu in a vial and desalt the solution using a standard protocol.

Next, prepare a two milligram per milliliter solution of two five dihydroxy benzoic acid in methanol, and mix two microliters of this solution with two microliters of the alluded phospho peptide solution. Inside a pipet tip spot this mixture directly onto the matrix assisted laser desorption ionization plate, and allow the spot to completely dry. Analyze the spots by matrix assisted laser Desorption ionization time of flight mass spectrometry finally regenerate the column by flushing it thoroughly with water, followed by methanol capillaries modified with multiple layers of available iron.

Three groups are ideal for use in immobilized metal ion affinity chromatography shown here are spectra obtained from digested proteins before and after enrichment, showing remarkable selectivity for 12 different phospho peptides. Representative characterization experiments provide valuable information about the emergence of new pores. Here is an image of a bulk powder monolith after 30 porous coordination polymer cycles known as PCP cycles using a scanning electron microscope.

After 30 PCP cycles, the size of the pore widths in the material was found to be less than three nanometers in diameter compared with the bulk polymer. The hybrid has a large amount of incremental area with small pores. This leads to an increase in nitrogen absorption through a range of pressures using Fourier transform infrared spectroscopy.

The initial incorporation of the carboxylic functional groups was visible as well as the growth of the coating after 10, 20 and 30 PCP cycles. Finally, thermo gravimetric analysis was used to measure the amount of additional metal sites that accumulated with each cycle. Shown here is gravimetric analysis through a range of temperatures, which confirms the presence of an increased number of metal sites with increased cycle number.

Following this protocol, a similar procedure can be developed for different metal centers in organic ligands in order to target efficient separations of different analytes based on their affinity for a particular framework. I believe that composite materials like ours that combine macroporous polymers with metal organic frameworks hold great promise in the field of separation science because they can vastly improve selectivity binding capacity and the overall efficiency of separations, both in analytical and preparative scale.