Evaluation of enzymatic digestion and liquid chromatography-mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin

A method for the complete peptide mapping of the model integral membrane protein bacteri-orhodopsin is demonstrated. Utilizing more effective enzymatic digestion, procedures with capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (MS/MS), all predicted tryptic digestion products were detected, as well as peptides from all previously reported post-translational modifications of bacteriorhodopsin. A significant contribution of chymotryptic-like digestion products was also observed. A characterization of the behavior of hydrophobic integral membrane peptides in a reversed-phase liquid chromatographic separation is also provided. The method reported here offers improved compatibility of the solubilizing reagents with both the chromatography and mass spectrometry, rendering it suitable for high-throughput proteomic applications.     

The Varietal Influence of Flavour Precursors from Grape Marc on Monoterpene and C13-Norisoprenoid Profiles in Wine as Determined by Membrane-Assisted Solvent Extraction (MASE) GC-MS

The winemaking by-product grape marc (syn. pomace) contains significant quantities of latent flavour in the form of flavour precursors which can be extracted and used to modulate the volatile composition of wine via chemical hydrolysis. Varietal differences in grapes are widely known with respect to their monoterpene content, and this work aimed to extend this knowledge into differences due to cultivar in volatiles derived from marc precursors following wine-like storage conditions. Marc extracts were produced from floral and non-floral grape lots on a laboratory-scale and from Muscat Gordo Blanco marc on a winery -scale, added to a base white wine for storage over five to six months, before being assessed using a newly developed membrane-assisted solvent extraction gas chromatography-mass spectrometry (GC-MS) method. The geraniol glucoside content of the marc extracts was higher than that of juices produced from each grape lot. In all wines with added marc extract from a floral variety, geraniol glucoside concentration increased by around 150–200%, with increases also observed for non-floral varieties. The relative volatile profile from extracts of the floral varieties was similar but had varied absolute concentrations. In summary, while varietally pure extracts would provide the greatest control over flavour outcomes when used in winemaking, aggregated marc parcels from floral cultivars may provide a mechanism to simplify the production logistics of latent flavour extracts for use in the wine sector.     

Fiber-optic infrared reflectance spectroelectrochemical studies of osmium and ruthenium nitrosyl porphyrins containing alkoxide and thiolate ligands

We have examined the redox behavior of the osmium and ruthenium compounds (OEP)M(NO)(OEt) and (OEP)M(NO)(SEt) (OEP = octaethylporphyrinato dianion; M = Os, Ru) by cyclic voltammetry and infrared spectroelectrochemistry. The compound (OEP)Os(NO)(OEt) undergoes a single reversible oxidation process in dichloromethane. In contrast, the thiolate compound (OEP)Os(NO)(SEt) undergoes a net irreversible oxidation resulting in formal loss of the SEt ligand. Extended Hückel calculations on crystal structures of these two compounds provide insight into the nature of their HOMOs. In the case of the alkoxide compound, the HOMO is largely metal centered, with 70% of the charge located in the metal's orbital and approximately 25% on the porphyrin ring. However, the HOMO of the thiolate compound consists of a pi bonding interaction between the metal dxz orbital and the px orbital on the sulfur, and a pi antibonding interaction between the metal d orbital and a pi* orbital on NO. The redox behavior of the Ru analogues have been determined, and are compared with those of the Os compounds.     

Self-assembled chiral superstructures composed of rigid achiral molecules and molecular scale chiral induction by dopants

We explore the phase behavior of a rigid achiral bent-core model system. Nematic and smectic phases form at higher densities, whereas micelles and columns composed of chiral clusters of these molecules self-assemble at lower densities. No nucleation mechanism requiring transient chirality is possible in the formation of these chiral superstructures due to the rigid achiral nature of the substituents. We show the chiral micelles are minima of the potential energy surface using energy minimization and parallel tempering simulations. Chiral dopants were found to induce the system to adopt a consistent chiral twist direction, the first molecular scale computer simulation of this effect.     

Proteomic characterization of plasma‐derived clotting factor VIII–von Willebrand factor concentrates

Proteomic methods were used to identify the levels of impurities in three commercial plasma‐derived clotting factor VIII‐von Willebrand factor (FVIII/VWF) concentrates. In all three concentrates, significant amounts of other plasma proteins were found. In Octanate and Haemoctin, two concentrates developed in the 1990s, the major impurities identified were inter‐α inhibitor proteins, fibrinogen and fibronectin. These two concentrates were also found to contain additional components such as clotting factor II (prothrombin) that are known activators of FVIII. In Wilate, a recently developed FVIII/VWF concentrate, the amount of these impurities was significantly reduced. Batch‐to‐batch variations and differences between three investigated products were detected using iTRAQ, an isotope labeling technique for comparative MS, demonstrating the potential value of this technique for quality control analysis. The importance of thorough proteomic investigations of therapeutic FVIII/VWF preparations from human plasma is also discussed.     

Unexpected nitrosyl-group bending in six-coordinate [M(NO)](6) sigma-bonded aryl(iron) and -(ruthenium) porphyrins.

The six-coordinate nitrosyl sigma-bonded aryl(iron) and -(ruthenium) porphyrin complexes (OEP)Fe(NO)(p-C(6)H(4)F) and (OEP)Ru(NO)(p-C(6)H(4)F) (OEP = octaethylporphyrinato dianion) have been synthesized and characterized. Single-crystal X-ray structure determinations reveal an unprecedented bending and tilting of the MNO group for both [MNO](6) species as well as significant lengthening of trans axial bond distances. In (OEP)Fe(NO)(p-C(6)H(4)F) the Fe-N-O angle is 157.4(2) degrees, the nitrosyl nitrogen atom is tilted off of the normal to the heme plane by 9.2 degrees, Fe-N(NO) = 1.728(2) A, and Fe-C(aryl) = 2.040(3) A. In (OEP)Ru(NO)(p-C(6)H(4)F) the Ru-N-O angle is 154.9(3) degrees, the nitrosyl nitrogen atom is tilted off of the heme normal by 10.8 degrees, Ru-N(NO) = 1.807(3) A, and Ru-C(aryl) = 2.111(3) A. We show that these structural features are intrinsic to the molecules and are imposed by the strongly sigma-donating aryl ligand trans to the nitrosyl. Density functional-based calculations reproduce the structural distortions observed in the parent (OEP)Fe(NO)(p-C(6)H(4)F) and, combined with the results of extended Hückel calculations, show that the observed bending and tilting of the FeNO group indeed represent a low-energy conformation. We have identified specific orbital interactions that favor the unexpected bending and tilting of the FeNO group. The aryl ligand also affects the Fe-NO pi-bonding as measured by infrared and (57)Fe M?ssbauer spectroscopies. The solid-state nitrosyl stretching frequencies for the iron complex (1791 cm(-)(1)) and the ruthenium complex (1773 cm(-)(1)) are significantly reduced compared to their respective [MNO](6) counterparts. The M?ssbauer data for (OEP)Fe(NO)(p-C(6)H(4)F) yield the quadrupole splitting parameter +0.57 mm/s and the isomer shift 0.14 mm/s at 4.2 K. The results of our study show, for the first time, that bent Fe-N-O linkages are possible in formally ferric nitrosyl porphyrins.     

Use of short monolithic columns for isolation of low abundance membrane proteins

Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.     

Proteomics as a tool for optimization of human plasma protein separation

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.