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 ...

Bahauddin Zakariya University

Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment.

Versatile nanocomposites in phosphoproteomics: a review.

The new synthetic polymers have a key role to play in the separation science. The derivatization of these polymers has made them an efficient class of substrate, having unique properties and the selectively tailored surface chemistries for target molecules. The deeper and detailed characterization of complex sample types has become feasible due to the enhanced selectivity and sensitivity offered by these polymer materials. In present work, a bifunctional monomer glycidyl propargyl ether (GPE) is thermally polymerized with divinylbenzene to form poly(GPE/DVB). Some of the physical and chemical properties are characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and Fourier transform infrared spectroscopy (FTIR). The synthesized polymer is further derivatized to IMAC (immobilized metal ion affinity chromatography) and is investigated by loading different metal ions (Fe(3+), Ti(4+), Zr(4+), and La(3+)). The trypsin-digested products of phosphoproteins, such as casein, nonfat milk, egg yolk, and human blood serum, are used to explore its phosphopeptide enrichment ability from complex samples followed by the off-line MALDI-MS analysis. Furthermore, polymeric reversed phase (RP) is created by octadecyl amine (ODA) to be employed in the desalting of complex mixtures and the results are compared with commercially available ZipTip C-18 and Aspire RP30 Desalting Tip. Serum profiling of healthy and diseased samples demonstrates the potential of this new polymer to impart in the disease diagnosis. Ovarian carcinoma serum samples are used for the detection of phosphopeptides based biomarkers.

High affinity phosphopeptides enrichment and desalting of biological materials on newly engineered poly(glycidyl propargyl ether/divinyl benzene).

The polymeric materials have contributed significantly in the area of bioanalytical science. The functionalization of polymeric backbone after its development brings unique selectivity towards the target biomolecules. In present work, the functionalities of choice have been introduced through the ring-opening of allyl glycidyl ether. The utility of polymer is widened through derivatizations to immobilized metal ion affinity chromatographic (IMAC) material for the phosphopeptides enrichment and Reversed Phase (C-18) for the desalting prior to MALDI-MS analysis. The polymer-IMAC in addition to Fe(3+) is also immobilized with lanthanide ions like La(3+), Eu(3+), and Er(3+). The amount of Fe(3+) immobilized is determined as 0.7928 mg/g. Spherical morphology with narrow particle size dispersion is revealed by scanning electron microscopy (SEM). The surface area, pore volume and size distribution is determined by nitrogen adsorption porosimetery. The elemental composition and purity level is confirmed by energy dispersive X-ray spectroscopy (EDX) data. The derivatization to IMAC and RP is evaluated by Fourier transform infrared (FT-IR) spectroscopy. The polymer enables the efficient phosphopeptide enrichment to equal degree from casein variants, non-fat milk, egg yolk, human serum, and HeLa cell extract. The identification of phosphorylation sites can lead to the phosphorylation pathways to understand the post-translational modifications. The identification with their sequence coverage is made using Mascot and Phosphosite Plus. It is sensitive to enrich the phosphopeptides down to 2 femtomoles with very high selectivity of 1:2000 with BSA background. These attributes are linked to the higher surface area (173.1554 m(2)/g) of the designed polymer. The non-specific bindings, particularly the Fe(3+) linked acidic residues are also avoided. Four characteristic phosphopeptides (fibrinopeptide A and their hydrolytic products) from fibrinogen α-chain are identified from the human serum after the enrichment, which have link to the hepatocellular carcinoma (HCC). The proportions of fibrinogen and their phosphorylation products enriched by poly(AGE/DVB)-IMAC open new horizons in the biomarker discovery.

Newly developed poly(allyl glycidyl ether/divinyl benzene) polymer for phosphopeptides enrichment and desalting of biofluids.

Determination of the availability of phases for specific separations is an important task achieved by a separation chemist. This becomes vital when the complex samples like biofluids are dealt with in proteome science. The work presented here involves the synthesis and application of terpolymeric sorbent with different functionalizations adopted for the selective enrichment of biomolecules of interest from biological fluids. Synthesis of terpolymer was carried out by the radical polymerization of monomers: methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross-linking agent, benzoyl peroxide as initiator and chloroform as a porogenic solvent. Characterization was done through Fourier transform infrared spectroscopy, scanning electron microscopy and nitrogen adsorption porosimetry. The polymer was further modified to immobilized metal ion affinity chromatographic material, with immobilized Fe(3+) /La(3+) ions that allowed phosphopeptide enrichment from tryptic digests of standard proteins as well as milk, egg yolk and human serum. Sensitivity of enrichment down to 50 fmol was achieved in the presence of complex protein background as bovine serum albumin. Hydrophobicity was introduced through octadecyl amine, which provides comparable results to ZipTip C18 /C4 for desalting of complex mixtures of all caseins. Analysis of the enriched content was performed by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). Copyright © 2014 John Wiley & Sons, Ltd.

Adeela Saeed

Institute of Chemical Sciences

Bahauddin Zakariya University

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

Adeela Saeed

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Chemical Sciences

Bahauddin Zakariya University

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

Institute of Chemical Sciences