Monolithic Poly(styrene-co-divinylbenzene) Columns for Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Polypeptide.

The organic polymer-based monolithic columns have been evaluated as the separation media for analysis of peptides using supercritical fluid chromatography-mass spectrometry (SFC-MS). We demonstrate for the first time the SFC-MS separation of a mixture of polypeptides carried out using poly(styrene-co-divinylbenzene) monolithic columns and carbon dioxide/methanol mobile phase. A gradient from 2 to 40% methanol modifier containing 0.1% TFA as an acidic additive was applied for the optimized elution and the separation was achieved in less than 3 min. Selected ion monitoring enabled detection of selected masses characteristic of three ionophoric pentadecapeptide antibiotics gramicidin A, B, and C and their two corresponding isoforms. Furthermore, their identity was confirmed through determination of their [M + 2H]2+, [M + 2Na]2+, and [M + H + Na]2+ ions acquired by positive-ion electrospray ionization-mass spectrometry (ESI-MS).

Liposome Artificial Membrane Permeability Assay by MALDI-hydrogen-deuterium exchange mass spectrometry for peptides and small proteins.

The pharmaceutical industry's focus has expanded to include peptide and protein-based therapeutics; however, some analytical challenges have arisen along the way, including the urgent need for fast and robust measurement of the membrane permeability of peptides and small proteins. In this study, a simple and efficient approach that utilizes MALDI-TOF-MS to study peptide and protein permeability through an artificial liposome membrane in conjunction with a differential hydrogen-deuterium exchange (HDX) methodology is described. A non-aqueous (aprotic) matrix was evaluated for use with MALDI sample preparation in order to eliminate undesirable hydrogen-deuterium back-exchange. Peptides and proteins were incubated with liposomes and their penetration into the liposome membrane over time was measured by MALDI-MS. A differential HDX approach was used to distinguish the peptides outside of the liposome from those inside. In this regard, the peptides on the outside of the liposomes were labeled using short exposure to deuterium oxide, while the peptides inside of the liposomes were protected from labeling. Subsequently, the unlabeled versus labeled peak area ratios for peptide and protein samples were compared using MALDI-TOF-MS. In this proof-of-concept study, we developed the Liposome Artificial Membrane Permeability Assay (LAMPA) workflow to study three well-known membrane-active model peptides (melittin, alamethicin, and gramicidin) and two model proteins (aprotinin and ubiquitin). The permeability results obtained from this were corroborated by previously reported data for studied peptides and proteins. The proposed LAMPA by MALDI-HDX-MS can be applied in an ultra-high-throughput manner for studying and rank-ordering membrane permeability of peptides and small proteins.

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer into hepatic cells by asialofetuin-labeled liposomes.

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta-Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF-liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF-liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta-Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2-fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner.

Colorimetric assay of gramicidin A in the presence of surfactants and phospholipids.

Gramicidin A, a hydrophobic polypeptide containing 4 tryptophan residues/molecule, may be determined quantitatively after reaction with 4-(dimethylamino)benzaldehyde, a method previously proposed for tryptophan analysis. The assay may be carried out even in the presence of various surfactants and phospholipids at high concentrations.

Size-exclusion high-performance liquid chromatography in the study of the autoassociating antibiotic gramicidin A in micellar milieu.

Gramicidin A (gA) is a polypeptide antibiotic which forms dimeric channels specific for monovalent cations in biological membranes. It is a polymorphic molecule that adopts several different conformations, double-stranded (ds) helical dimers (pore conformation) and single-stranded beta-helical dimers (channel conformation). This study investigated the conformational adaptability of gramicidin A when incorporated into micelles as membrane-mimetic model system. Taking advantage of our reported, versatile, size-exclusion high-performance liquid chromatography (SE-HPLC) strategy that allows the separation of double-stranded dimers and monomers, we have quantitatively characterized the conformational transition undergone by the peptide in the micellar milieu. The importance of both hydrophobic/hydrophilic moieties of the amphipaths in the stabilization of concrete conformational species is demonstrated using detergents with different hydrocarbon chain length and/or polar head. SE-HPLC is a valuable, rapid, accurate technique for the structural characterization of hydrophobic autoassociating peptides that work in lipid environments such as biological membranes.

Salivary levels of gramicidin after use of a tyrothricin-containing gargle/mouth-wash and tyrothricin lozenges.

Three pharmaceutical preparations for the disinfection of the oropharynx were tested with regard to the gramicidin concentration obtained in the saliva after their appropriate application. Peak values followed a dose-concentration relationship and were highest after sucking a lozenge containing 10 mg tyrothricin (mean 109.3 mg/L) followed by that of a gargle/mouth-wash containing 667 mg tyrothricin/L (mean 21.1 mg/L) and of a lozenge containing 4 mg tyrothricin (mean 14.4 mg/L).

Screening for small molecules' bilayer-modifying potential using a gramicidin-based fluorescence assay.

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides a possible mechanism for "off-target" drug effects. We have previously shown that channels formed by the linear gramicidins are suitable probes for changes in lipid bilayer properties, as experienced by bilayer-spanning proteins. We now report a gramicidin-based fluorescence assay for changes in bilayer properties. The assay is based on measuring the time course of fluorescence quenching in fluorophore-loaded large unilamellar vesicles, due to entry of a gramicidin channel-permeable quencher. The method is scalable and suitable for both mechanistic studies and high-throughput screening for bilayer-perturbing, potential off-target effects, which we illustrate using capsaicin (Cap) and other compounds.

TOF-SIMS analysis using C60. Effect of impact energy on yield and damage.

C60 has been shown to give increased sputter yields and, hence, secondary ions when used as a primary particle in SIMS analysis. In addition, for many samples, there is also a reduction in damage accumulation following continued bombardment with the ion beam. In this paper, we report a study of the impact energy (up to 120 keV) of C60 on the secondary ion yield from a number of samples with consideration of any variation in yield response over mass ranges up to m/z 2000. Although increased impact energy is expected to produce a corresponding increase in sputter yield/rate, it is important to investigate any increase in sample damage with increasing energy and, hence, efficiency of the ion beams. On our test samples including a metal, along with organic samples, there is a general increase in secondary ion yield of high-mass species with increasing impact energy. A corresponding reduction in the formation of low-mass fragments is also observed. Depth profiling of organic samples demonstrates that when using C60, there does not appear to be any increase in damage evident in the mass spectra as the impact energy is increased.

Improvements in instrumentation for thermospray operation on a magnetic sector mass spectrometer.

Changes in the design of a thermospray liquid chromatography-mass spectrometry ion source can be made to improve the sensitivity towards solute related ions and therefore extend the practical utility of the complete system. The addition of a discharge ionization facility provides much greater scope for gradient elution analyses and forms the basis of a method which offers increased structural information. All of these changes are illustrated by practical examples.

New high-performance liquid chromatography-based methodology for monitoring the conformational transitions of self-associating hydrophobic peptides, incorporated into liposomes.

A new high-performance size-exclusion chromatographic strategy is reported for the analysis of the hydrophobic self-associating peptide gramicidin A, incorporated into artificial phospholipid vesicles (liposomes). The method is based on the direct injection of a few microlitres of the gramicidin A-containing liposome suspension into the column, which is eluted with a non-polar solvent, such as tetrahydrofuran. The type and amount of information which can be derived from this methodology have been evaluated. Using this chromatographic approach, a correlation has been unambiguously shown to exist between the organization of the peptide in the vesicles and a number of variables involved in the method of preparation of liposomes. Finally, a gramicidin A conformational transition has been monitored in the phospholipid vesicles which proved to be dependent on the class of phospholipid present in the liposome.

Micro-electrospray with stainless steel emitters.

The physical processes underlying micro-electrospray (micro-ES) performance were investigated using a stainless steel (SS) emitter with a blunt tip. Sheathless micro-ES could be generated at a blunt SS tip without any tapering or sanding if ESI conditions were optimized. The Taylor cone was found to shrink around the inner diameter of the SS tubing, which permitted a low flow rate of 150 nL/min for sheathless microspray on the blunt tip (100 microm i.d. x 400 microm o.d.). It is believed that the wettability and/or hydrophobicity of SS tips are responsible for their micro-ES performance. The outlet orifice was further nipped to reduce the size of the spray cone and limit the flow rate to 50-150 nL/min, resulting in peptide detection down to attomole quantities consumed per spectrum. The SS emitter was also integrated into a polymethylmethacrylate microchip and demonstrated satisfactory performance in the analysis and identification of a myoglobin digest.