Reinhard I. Boysen

Hydrophilic interaction chromatography coupled to electrospray mass spectrometry for the separation of peptides and protein digests.

In this study, the analysis of a peptide set, chosen for their differences in hydrophilicity, and the tryptic digests of bovine cytochrome c and beta-lactoglobulin by hydrophilic interaction chromatography-electrospray ionisation mass spectrometry (HILIC-ESI-MS) is demonstrated. Two different types of HILIC phases, i.e., an amide- and an amino-modified silica-based phase, packed into narrow bore or capillary columns, were investigated with separations conducted under either low pH or neutral pH conditions. The separation performance of the two HILIC columns with respect to peak efficiency and selectivity have been documented under these different mobile phase conditions, and the results compared with the performance of a conventional capillary reversed-phase C18 column of similar dimensions. It was found that very good separation of the peptide set could be achieved by using the amide-modified silica column over a broad pH range. Moreover, with the protein digest samples, excellent separation of the tryptic digests was obtained with the amide-modified HILIC column under neutral pH conditions. Compared to the conventional reversed-phase C18 separations, the use of these HILIC columns not only provided complementary separation selectivity, but also offered the capability to identify unique peptides using tandem HILIC-mass spectrometric techniques. These studies therefore highlight the potential of HILIC procedures for future proteomic applications.

Aqueous normal-phase retention of nucleotides on silica hydride columns

The use of silica hydride-based stationary phases for the retention and analysis of nucleotides has been investigated. Both reversed-phase columns with a hydride surface underneath as well as those with an unmodified or a minimally modified hydride material were tested. With these systems, an aqueous normal-phase mode was used with high organic content mobile phases in combination with an additive to control pH for the retention of the hydrophilic nucleotides. Isocratic and gradient elution formats have been used to optimize separations for mixtures containing up to seven components. All conditions developed are suitable for methods that utilize mass spectrometry detection.

Rapid solid-phase extraction and analysis of resveratrol and other polyphenols in red wine

Red wine has long been credited as a good source of health-beneficial antioxidants, including the bioactive polyphenols catechin, quercetin, and (E)-resveratrol. In this paper, we report the application of reusable molecularly imprinted polymers (MIPs) for the selective and robust solid-phase extraction (SPE) and rapid analysis of (E)-resveratrol (LOD=8.87×10(-3) mg/L, LOQ=2.94×10(-2) mg/L), along with a range of other polyphenols from an Australian Pinot noir red wine. Optimization of the molecularly imprinted solid-phase extraction (MISPE) protocol resulted in the significant enrichment of (E)-resveratrol and several structurally related polyphenols. These secondary metabolites were subsequently identified by RP-HPLC and μLC-ESI ion trap MS/MS methods. The developed MISPE protocol employed low volumes of environmentally benign solvents selected according to the Green Chemistry principles, and resulted in the recovery of 99% of the total (E)-resveratrol present. These results further demonstrate the potential of generic protocols for the analysis of target compound with health beneficial properties within the food and nutraceutical industries using tailor-made MIPs.

Peptide mapping by reversed-phase high-performance liquid chromatography employing silica rod monoliths.

In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.

Electrochromatographic Characterization of Etched Chemically-Modified Capillaries with Small Synthetic Peptides

In this investigation, various capillary electrochromatographic (CEC) experiments have been employed to characterize the properties of etched, chemically-modified surfaces of open tubular capillary columns with peptides as solute probes and under conditions of variable voltage, temperature and solvent composition. The separation performance of etched capillaries with either n-octadecyl or liquid crystal moieties derived from a cholesterol phase bonded to the surface were compared. With the liquid crystal bonded species, interesting and significantly different variations in retention behavior of peptides are obtained compared to those observed with the corresponding n-octadecyl modified surfaces by changes in temperature, solvent composition and field strength. These peptide separations illustrate the usefulness of this CEC approach for practical applications, where both the retention characteristics of the charged analytes as well as the selectivity differences due to the surface properties of the etched chemically-modified surfaces of open tubular capillary columns can be rationally modulated. As in HPLC, appropriate choice of CEC experimental variables, including the chemical properties of the immobilized ligand(s), represents a powerful tool for optimizing resolution.

Open Tubular‐Capillary Electrochromatography: Developments and Applications from 2013 to 2015

Open tubular CEC (OT‐CEC) separates analyte mixtures by a combination of electrophoretic, electro‐osmotic, and/or chromatographic effects. OT‐CEC research is an active and growing field, with studies encompassing a wide range of investigations related to new strategies for chemical modification of the inner surface of the capillary, leading to the introduction of novel stationary phase coatings. This review has examined the literature on OT‐CEC from 2013 to August 2015 and highlights the developments in the fabrication of highly selective stationary phases, based on materials that include cyclodextrin chiral selectors, graphene and graphene oxide, metal‐organic frameworks, molecularly imprinted polymers, nanoparticles, nanolatex particles, nanocomposites, in situ generated polymers, block polymers, tentacle‐type polymers, polyelectrolyte multilayers, polysaccharides, phospholipids, and proteins. This review, while considering the development of novel OT‐CEC coating materials, specifically examines different immobilization or coating methodologies and approaches and also discusses the separation mechanisms that occur with these new materials. These OT‐CEC coatings are intended mainly to separate low molecular weight molecules relevant to the pharmaceutical, agricultural, and food industries as well as for use in environmental monitoring.

Preparation of molecularly imprinted polymers for the selective recognition of the bioactive polyphenol, (E)-resveratrol

(E)-Resveratrol imprinted polymers have been rationally designed with the aid of molecular modelling and NMR spectroscopic titration techniques to determine the optimal ratio of the template to functional monomer for polymer formation. Based on this approach, (E)-resveratrol imprinted polymers were prepared via non-covalent self-assembly with the functional monomer 4-vinylpyridine (4VP) in a 1:3 molar ratio. Polymerisation in the presence of a cross-linker resulted in rigid block copolymers that had selective capacities towards (E)-resveratrol (e.g. 14 μmol/g) when compared to the non-imprinted reference polymer. The selectivity of these MIPs was also examined using several structurally related polyphenolic compounds to determine the influence of polyphenolic hydroxyl number and position on binding and molecular recognition.

Enrichment of (E)-Resveratrol from Peanut Byproduct with Molecularly Imprinted Polymers

Molecularly imprinted solid phase extraction (MISPE) has been employed to isolate and concentrate bioactive polyphenols from peanut press waste. To this end, a molecularly imprinted polymer (MIP) templated with the phytoalexin (E)-resveratrol has been prepared via self-assembly with the functional monomer 4-vinylpyridine (4VP) in a 1:3 molar ratio. Subsequent molecular interrogation of the MIP binding sites demonstrated preferential structural selectivity for (E)-resveratrol with respect to other structurally related naturally occurring compounds. This selectivity was subsequently exploited to achieve substantial sample cleanup of peanut press waste under aqueous conditions with significant enrichment of (E)-resveratrol (>60 fold) requiring minimal sample preparation.

Molecular Identification of Lyso-Glycerophosphocholines as Endogenous Immunosuppressives in Bovine and Rat Gonadal Fluids

Abstract The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.

Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions

The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5-9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides.