Insights into chemoselectivity principles in metal oxide affinity chromatography using tailored nanocast metal oxide microspheres and mass spectrometry-based phosphoproteomics.

The ability to comprehensively characterize biological samples, including tissues and body fluids, opens up new possibilities to diagnose and treat diseases and to better understand fundamental biological processes. For this purpose, suitable experimental workflows need to be designed. In this context, materials with particular chemoselective properties are used for the enrichment of certain classes of (bio)molecules. Metal oxides such as titanium dioxide have become the materials of choice for the large-scale study of protein phosphorylation in phosphoproteomics. Despite their widespread use, the main factors influencing their performance (for example, affinity and specificity) are not completely understood. This understanding is, however, crucial to develop improved materials and methods. Here, we used the nanocasting method to prepare microspheres of seven metal oxides with comparable textural properties, allowing an objective comparison of the materials and their binding properties. We evaluated these materials with samples of different complexity, ranging from synthetic peptides to whole cell lysates, using liquid chromatography-tandem mass spectrometry as a readout. A set of more than 7000 identified phosphopeptides allowed us to study differences between the metal oxide sorbents in detail. Importantly, the performance of the affinity materials was found to be mainly correlated with the oxides' isoelectric points (IEPs), with the materials that enriched the highest number of phosphopeptides having an IEP of around 6. This included the widely used TiO2 and ZrO2, but also In2O3 that was not previously known to possess affinity to phosphates. This finding supports the conclusion that the IEP has a stronger influence than the particular type of metal oxide and contrasts earlier reports that compared a limited number of materials with often unknown textural properties. Taken together, we introduce new metal oxides suitable for phosphopeptide enrichment, provide deeper insight into the properties that affect their performance, and offer a strategy to optimize enrichment protocols and materials.

Comparison of different amino-functionalization procedures on a selection of metal oxide microparticles: degree of modification and hydrolytic stability.

Amino-modified metal oxide materials are essential in a wide range of applications, including chromatography, ion adsorption, and as biomaterials. The aim of this study is to compare different functionalization techniques on a selection of metal oxides (SiO(2), TiO(2), ZrO(2), and SnO(2)) in order to determine which combination has the optimal properties for a certain application. We have used the nanocasting approach to synthesize micrometer-sized TiO(2), ZrO(2), and SnO(2) particles, which have similar morphologies and porosities as the starting mesoporous SiO(2) microparticles (Lichroprep Si 60). These metal oxides were subsequently functionalized by four different approaches, (a) covalent bonding of 3-aminopropyltriethoxysilane (APTES), (b) adsorption of 2-aminoethyl dihydrogen phosphate (AEDP), (c) surface polymerization of aziridine (AZ), and (d) electrostatic interaction of poly(ethylenimine) (PEI), to produce a high surface coverage of amino groups on their surfaces. Scanning electron microscopy, nitrogen physisorption, and X-ray diffraction were used to characterize the unmodified metal oxide particles, while thermogravimetric analysis, ninhydrin adsorption, and ζ potential titrations were applied to gain insight into the successfulness of the various surface modifications. Finally, the hydrolytic stability at pH 2 and 10 was investigated by ζ potential measurements. Unfortunately, the AEDP approach was not able to produce efficient amino-modification on any of the tested metal oxide surfaces. On the other hand, modifications with APTES, aziridine, and PEI appeared to give fairly stable amino-functionalizations at high pH values for all metal oxides, while these modifications were easily detached at pH 2, with the exception of SnO(2), where the AZ and PEI samples were stable up to 40 h. The results are expected to give valuable insights into the possibility of replacing amino-modified silica with more hydrolytically stable metal oxides in various application fields, for example, chromatography and drug delivery.

Modified zeolitic imidazolate framework-8 as solid-phase microextraction Arrow coating for sampling of amines in wastewater and food samples followed by gas chromatography-mass spectrometry.

In this study, a novel solid phase microextration (SPME) Arrow was prepared for the sampling of volatile low molecular weight alkylamines (trimethylamine (TMA) and triethylamine (TEA)) in wastewater, salmon and mushroom samples before gas chromatographic separation with mass spectrometer as detector. Acidified zeolitic imidazolate framework-8 (A-ZIF-8) was utilized as adsorbent and poly(vinyl chloride) (PVC) as the adhesive. The custom SPME Arrow was fabricated via a physical adhesion: (1) ZIF-8 particles were suspended in a mixture of tetrahydrofuran (THF) and PVC to form a homogeneous suspension, (2) a non-coated stainless steel SPME Arrow was dipped in the ZIF-8/PVC suspension for several times to obtain a uniform and thick coating, (3) the pore size of ZIF-8 was modified by headspace exposure to hydrochloric acid in order to increase the extraction efficiency for amines. The effect of ZIF-8 concentration in PVC solution, dipping cycles and aging temperature on extraction efficiency was investigated. In addition, sampling parameters such as NaCl concentration, sample volume, extraction time, potassium hydroxide concentration, desorption temperature and desorption time were optimized. The Arrow-to-Arrow reproducibilities (RSDs) for five ZIF-8 coated Arrows were 15.6% and 13.3% for TMA and TEA, respectively. The extraction with A-ZIF-8/PVC Arrow was highly reproducible for at least 130 cycles without noticeable decrease of performance (RSD<12.5%). Headspace SPME of 7.5mL sample solution with the fabricated ZIF-8 coated Arrow achieved linear ranges of 1-200ngmL-1 for both TMA and TEA. The limit of quantitation (LOQ) was 1ngmL-1 for both TMA and TEA. The method was successfully applied to the determination of TMA and TEA in wastewater, salmon and mushroom samples giving satisfactory selectivity towards the studied amines.

Phospholipids covalently attached to silica particles as stationary phase in nano-liquid chromatography.

Silica particles were covalently modified with phospholipids and used as packing material for nano-liquid chromatography (nano-LC). This modification involved aminopropylsilylation of the raw silica particles using 3-(aminopropyl)-triethoxysilane, covalent binding of glutaraldehyde molecules to the aminopropylsilylated particles, and finally covalent binding of different phospholipid vesicles containing primary amino groups to the iminoaldehyde silica particles. Capillaries with an inner diameter of 100µm were packed with phospholipid-coated silica particles using a slurry packing method. The packed capillaries were tested in nano-LC with UV-detection for the separation of acidic, neutral, and basic model analytes. The effect of the buffer ion on the retention factor of the analytes was evaluated using buffer solutions with constant ionic strength and pH. In addition, the effect of the volume of methanol in the mobile phase was studied. The calculated distribution coefficients (logK(D)) of the model compounds were in agreement with those reported in the literature. A good correlation between logK(D) values and octanol/water partitioning coefficients (P(o/w)) for neutral hydrophobic analytes was obtained proving the applicability of the method for predicting partitioning of the compounds with the biomembranes.

Polyethylenimine-modified metal oxides for fabrication of packed capillary columns for capillary electrochromatography and capillary liquid chromatography.

The need for novel packing materials in both capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) is apparent and the development towards more selective, application-oriented chromatographic phases is under progress world-wide. In this study we have synthesized new polyethyleneimine (PEI) functionalized Mn(2)O(3), SiO(2), SnO(2), and ZrO(2) particles for the fabrication of packed capillary columns for CEC and CLC. The nanocasting approach was successful for the preparation of functionalized metal oxide materials with a controlled porosity and morphology. PEI functionalization was done using ethyleneimine monomers to create particles which are positively charged in aqueous solution below pH 9. This functionalization allowed the possibility to have both hydrophobic (due to its alkyl chain) and ionic interactions (due to positively charged amino groups) with selected compounds. For comparison aminopropyl-functionalized silica was also synthesized and tested. Both slurry pressure and electrokinetic packing procedures used gave similar results, but fast sedimentation of the material caused some problems during the packing. The high stability and wide pH range of PEI-functionalized SiO(2) material, with potential for hydrophobic and electrostatic interactions, proved to be useful for the CEC and CLC separation of some model acidic and neutral compounds.