Evaluation of migration behaviour of therapeutic peptide hormones in capillary electrophoresis using polybrene-coated capillaries

Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and for rapid selection of the optimum pH for separation of mixtures of the modelled compounds. In this work, equations describing the effect of pH on electrophoretic behaviour were used to investigate migration of a series of polyprotic amphoteric peptide hormones between pH 2 and 12 in polybrene-coated capillaries. Polybrene (hexadimethrin bromide) is a polymer composed of quaternary amines that is strongly adsorbed by the fused-silica inner surface, preventing undesired interactions between the peptides and the inner capillary wall. In polybrene-coated capillaries the separation voltage must be reversed, because of the anodic electroosmotic flow promoted by the polycationic polymer attached to the inner capillary wall. The possibility of using polybrene-coated capillaries for determination of accurate ionization constants has been evaluated and the optimum pH for separation of a mixture of the peptide hormones studied has been selected. Advantages and disadvantages of using bare fused-silica and polybrene-coated capillaries for these purposes are discussed.     

Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn‐superoxide dismutase

The native form of Cu,Zn‐superoxide dismutase (SOD‐1) is a homodimer that coordinates one Cu²⁺ and one Zn²⁺ per monomer. Cu²⁺ and Zn²⁺ ions play crucial roles in enzyme activity and structural stability, respectively. In addition, dimer formation is essential for SOD‐1 functionality, and in humans several SOD‐1 mutant isoforms have been associated with certain types of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder. In this paper we used capillary electrophoresis and mass spectrometry to study the different structures of bovine SOD‐1. The metal ions of the native enzyme (Cu₂,Zn₂‐dimer SOD‐1) were released in acidic medium in order to obtain apo‐SOD‐1, which is a monomer. Both substances were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and capillary electrophoresis with ultraviolet and electrospray ionization mass spectrometry detection (CE/UV and CE/ESI‐MS, respectively). With MALDI‐TOF‐MS, using matrices of sinapinic acid (SA) or 2,5‐dihydroxybenzoic acid (DHB) with or without trifluoroacetic acid (TFA), similar mass spectra were obtained for the metalated and non‐metalated samples. In both cases, an average molecular mass corresponding to the apo‐monomer SOD‐1 was calculated. This finding indicated that the metals were released from the Cu₂,Zn₂‐dimer SOD‐1 during sample preparation or ionization. For CE/UV and CE/ESI‐MS, two background electrolytes (BGEs) potentially compatible with ESI‐MS detection were used, namely 1 M of acetic acid (pH 2.3) and 10 mM of ammonium acetate (pH 7.3). Using a sheath liquid of 2‐propanol/water (60:40 v/v), with or without 0.1% v/v of formic acid, CE/ESI‐MS sensitivity was enhanced when the acidic BGE and the acidic sheath liquid were used. However, the electrophoretic profiles and the mass spectra obtained suggested that the metals of Cu₂,Zn₂‐dimer SOD‐1 were released, which generated the apo‐monomer during the electrophoretic separation. The neutral BGE provided enhanced conditions for the detection of the native enzyme. The differences between the mass spectra obtained for the Cu₂,Zn₂‐dimer and the apo‐monomer forms were significant and the presence of formic acid in the sheath liquid affected only sensitivity. Our results highlight the importance of selecting appropriate non‐denaturing separation and detection conditions to obtain reliable structural information about non‐covalent protein complexes by CE/ESI‐MS. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on‐line solid‐phase extraction capillary electrophoresis‐mass spectrometry

Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β‐protein (Aβ) (Aβ(1–15) and Aβ(10–20) peptides) by on‐line immobilized metal affinity SPE‐CE (IMA‐SPE‐CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25‐fold and 5‐fold decrease in the LODs by IMA‐SPE‐CE‐UV for Aβ(1–15) and Aβ(10–20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE‐UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA‐SPE‐CE‐MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10–20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10–20) peptide was good in a narrow concentration range (0.25–2.5 μg/mL, R² = 0.93). Lastly, the potential of the optimized Ni(II)‐IMA‐SPE‐CE‐MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples.     

Capillary electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry for analysis of the novel erythropoiesis-stimulating protein (NESP)

NESP (novel erythropoiesis-stimulating protein) is a recently approved hyperglycosylated analogue of human erythropoietin (EPO) with a long-lasting effect. In this work, the capillary electrophoresis (CE) methodology proposed by the European Pharmacopoeia for the separation of EPO glycoforms has been modified for the separation of NESP glycoforms. Optimization of pH of the separation electrolyte has been fundamental in order to achieve baseline resolution of seven peaks corresponding to NESP glycoforms. Intact NESP has also been characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). An accurate approximation to an average molecular mass of the NESP molecule has been obtained, taking into account the strong influence of laser intensity upon the MALDI-TOF mass spectra found.     

Thermotropic liquid crystal polyesters derived from 4,4′-biphenyldicarboxylic acid and oxyalkylene spacers

Several polyesters derived from bibenzoic acid and oxyalkylene glycols have been prepared and the ability of formation of mesophases was studied. The phase behaviour was investigated by means of differential scanning calorimetry, wide-angle X-ray diffraction and real-time variable-temperature small-angle X-ray diffraction. A common feature of all the studied polyesters, with ether groups in the spacer, is the inhibited ability for developing three-dimensional structures, in contrast to the polymers with all-methylene spacers. Moreover, the latter polymers show a monotropic mesophase behaviour but the former exhibit an enantiotropic transformation. Smectic structures of different types have been detected: The polyester derived from 3,3'-oxybis(1-propanol) produces a smectic S_A structure, while the polyester from triethylene glycol leads to a smectic S_C mesophase. The influence of the spacers on the dynamic mechanical relaxations of these polybibenzoates has been studied and discussed.     

Evaluation of fritless solid‐phase extraction coupled on‐line with capillary electrophoresis‐mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid

Fritless SPE on‐line coupled to CE with UV and MS detection (SPE‐CE‐UV and SPE‐CE‐MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C₁₈ sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE‐CE‐UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine‐enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro‐organic solution. Using SPE‐CE‐MS, peak area and migration time repeatabilities for the three opioid peptides were 12–27% and 4–5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE‐MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.     

Ionic liquid matrices for MALDI-TOF-MS analysis of intact glycoproteins

2,5-Dihydroxybenzoic acid (DHB) has been demonstrated to be a more suitable matrix than 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) to obtain reliable molecular mass values of intact glycoproteins because it prevents sugar fragmentation. Lack of spot homogeneity during the crystallization step was prevented by drying the sample-matrix mixture under vacuum conditions. Nevertheless, this sample-matrix preparation procedure requires a specific experimental setup and may be time-consuming. In this work, we investigated the effectiveness of different ionic liquid matrices (ILMs) with SA and DHB on the ionization of a set of intact glycoproteins with several degrees of glycosylation. The obtained results demonstrate that some of the tested ILMs allow detection of the studied intact glycoproteins. Furthermore, the selected optimum conditions solve the reproducibility issue of using the DHB as a solid matrix without the vacuum drying method and, surprisingly, avoid sugar fragmentation when both SA and DHB were used as ILMs.     

Migration behavior of therapeutic peptide hormones: prediction of optimal separation by capillary electrophoresis

A general equation that relates electrophoretic mobility of polyprotic peptide substances and pH of the running electrolytes is established, taking into account the species in solution and the activity coefficients. Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and selection of the optimum pH for separation of mixtures of the modelled compounds. The proposed relationships allow an important reduction of the experimental data needed for development of new separation methods. The accuracy of the proposed equations is verified by modelling the migration behavior of a heterogeneous series of polyprotic amphoteric peptide hormones. By calculating the values of predicted resolutions, selection of the optimum pH to perform separation of their mixtures becomes a rapid and simple process.     

Nanostructure reorganization in a thermotropic copolyester. A simultaneous WAXS and SAXS study

Thermally induced Angstrom and nanometer‐scale reorganization in thermotropic liquid crystalline polymer based on (1,4)‐hydroxybenzoic acid (B) and (2,6)‐hydroxynaphthoic acid (N) was investigated by simultaneous wide‐angle and small‐angle X‐ray scattering (SAXS, respectively). Extruded tapes 50 µm thick were annealed at 240°C under dry air conditions. The as‐received tape exhibited fiber‐like structure with crystalline order, whereas the SAXS patterns exhibited diamond‐shaped diffuse scattering elongated along the equatorial axis elucidating nanovoid morphology oriented along the extrusion axis. Guinier analyses showed that the radius of gyration R_g of nanovoids were ca. 17 nm along the extrusion axis. Heat treatment produced a sharpening of the 002 meridional reflection and the 110 equatorial reflection suggesting an improvement of molecular register and packing. The molecular alignment, as quantified by the order parameter , increased as well as the degree of crystallinity χ. On the other hand, SAXS intensity along the equatorial axis decreased evidencing reduction of R_g, i.e. lateral compression of the nanovoids and better molecular packing. Thermal treatment increased the thermal stability and the uniaxial tensile Young's modulus, E, along extrusion axis. However, the tapes exhibited microhardness anisotropy and the indentation anisotropy, ?H, gradually decreased suggesting reduction of elastic recovery in the molecular chain direction. Scanning electron microscopy evidenced an outer skin with an internal layered morphology that transformed into sheet‐like morphology with meandering fibrils. This investigation evidenced microstructure and morphology reorganization correlating with improved thermal and mechanical properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrokinetic and surface chemical characterizations of an irradiated microfiltration polysulfone membrane: comparison of two irradiation doses

The effect of ionizing radiation on the surface and electrokinetic characteristic parameters for a porous membrane of pore size 0.2 mum is determined and correlated with the irradiation dose (10 and 80 J/kg). Changes in NaCl permeability and membrane system electrical resistance determined from diffusion and impedance spectroscopy measurements are consistent with the increase of membrane pore radii/porosity, in agreement with SEM micrographs and reported results. Low irradiation dose seems to clean the membrane surface of impurities, according to XPS results, but the increase of irradiation doses could affect surface roughness. Due to the relatively high pore radius, ion transport numbers are practically independent of radiation and dose, but irradiation slightly modifies the membrane solution interface by increasing its weakly electronegative character, which could be of interest in the ultrafiltration of proteins or macromolecules.