Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane

Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.     

Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins

A comprehensive two-dimensional (2-D) liquid chromatographic separation system is presented. The system uses a microbore cation exchange column, operated under gradient conditions, as the first dimension separation. Effluent from this first column alternately fills one of two loops on a computer-controlled eight-port valve. A second pump then forces loop material onto a second column, a size exclusion column. UV detection is used, and the system is applied to the separation of protein standards and serum proteins. The 2-D system has a higher resolving power and peak capacity than either of the two columns used alone. The entire first column effluent is analyzed on the second column in virtually the same time it takes to complete the first column separation, without the use of stopped flow methods. The entire system is automated and operated under computer control. Three-dimensional (3-D) data representation provides a means of viewing peak profiles in either separation dimension and contour mapping of the 3-D data provides a more reliable means of peak identification from run to run than that provided by single-column elution times.     

Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using high-performance liquid chromatography with photolytic electrochemical detection

Aromatic amino acids, sulfur-containing amino acids, peptides containing such constituents, and proteins can now be detected in high-performance liquid chromatography by the use of on-line, postcolumn, continuous photolytic derivatization with electrochemical (HPLC-hv-EC) detection. The overall approach is a very simple, reproducible, rapid, and fully automatable approach for the determination of certain amino acids, peptides, and proteins with excellent selectivity, sensitivity, and linearities of response. Dual-electrode response ratios, lamp-on/lamp-off behavior, and chromatographic capacity factors all contribute to the enhanced selectivity of the overall HPLC-hv-EC determination for these particular classes of bioorganics and biopolymers. The analytical figures of merit, chromatography detection, and method validation approaches have all been optimally derived and demonstrated reproducible. Applications of the basic methodology to real-world samples are demonstrated and validated.     

Determination of boswellic acids in brain and plasma by high-performance liquid chromatography/tandem mass spectrometry

Peritumoral edema, one of the major causes for neurological disorders in brain tumor patients, is mainly treated with steroids, which unfortunately have significant side effects and interfere with the efficacy of chemotherapy. Boswellic acids, the main active ingredients of Boswellia serrata, are antiinflammatory agents, inhibiting 5-lipoxygenase, the key enzyme of leukotriene biosynthesis and one of the pathophysiological mechanisms of peritumoral edema. Based on positive results in clinical trials and animal studies, B. serrata resin dry extract was designated an orphan drug by the European Commission for the treatment of peritumoral edema resulting from brain tumors. Thus boswellic acids may be alternative drugs to corticosteroids. However, the question of the availability of boswellic acids in brain has not been addressed until now. Accordingly, a highly sensitive LC/MS method has been developed for the simultaneous determination of KBA and AKBA, the most potent boswellic acids, in plasma and brain. This method involves matrix-assisted liquid-liquid extraction on Extrelut NT followed by separation on reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using atmospheric pressure chemical ionization. Excellent linearity was obtained for the entire calibration range from 5 to 1500 ng/mL KBA and AKBA in plasma and 5 to 1000 ng/mL KBA and AKBA in brain. Validation assays of the lower limit of quantification as well as for the intra- and interday precision and accuracy met the international acceptance criteria for bioanalytical method validation. Moreover, the interchangeability of calibration curves generated in pork and rat brain homogenates could be demonstrated. Using the developed analytical method, KBA and AKBA could be detected for the first time in brain up to a concentration of 99 and 95 ng/g of brain, respectively, 3 h after the single oral administration of 240 mg/kg of dry B. serrata resin extract to Wistar rats. The developed method represents an appropriate tool to further study the time-dependent distribution of KBA and AKBA in plasma and brain as well as the absolute brain concentration after multiple doses and contributes thus to the optimization of the dosage regimen and to a better understanding of the therapeutic effects of B. serrata.     

Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis

The design, fabrication, and operation of a radial capillary array electrophoresis microplate and scanner for high-throughput DNA analysis is presented. The microplate consists of a central common anode reservoir coupled to 96 separate microfabricated separation channels connected to sample injectors on the perimeter of the 10-cm-diameter wafer. Detection is accomplished by a laser-excited rotary confocal scanner with four color detection channels. Loading of 96 samples in parallel is achieved using a pressurized capillary array system. High-quality separations of 96 pBR322 restriction digest samples are achieved in < 120 s with the microplate system. The practical utility and multicolor detection capability is demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel using a noncovalent 2-color staining method. This work establishes the feasibility of performing high-throughput genotyping separations with capillary array electrophoresis microplates.     

Microfabricated electrochemical detector for high-performance liquid chromatography

A microfabricated electrochemical cell has been developed as a disposable detector for flow injection analysis (FIA) and high-performance liquid chromatography (HPLC). The simplicity of the fabrication process allows this detector to be used as a low-cost, disposable device that can be replaced easily if its performance degrades rather than disassembling the detector and polishing the electrode surface, which is the usual procedure. The detector consists of thin film-metal electrodes-platinum working electrode, platinum auxiliary electrode, and silver/silver chloride coated on Pt reference electrode-deposited on a polyimide substrate with a locking layer of chromium in between. A microfluidic cover made of polyimide directs the solution flow across the electrodes. The detector was evaluated with FIA of ferrocyanide and HPLC of ascorbic acid and acetaminophen and a mixture of two pharmaceutical compounds-dextrorphan and levallorphan-with acetaminophen internal standard. The HPLC calibration curves showed good linearity (R(2) > 0.99). Limits of detection were 1 nM for acetaminophen, 1 nM for ascorbic acid, 50 nM for dextrorphan, and 80 nM for levallorphan. When detected with a commercial detector dextrorphan and levallorphan had lower limits of detection, 3 and 5 nM, respectively. Chromatograms of the mixture were comparable to those obtained with a commercial detector. The detector could be used continuously for about 48 h with FIA and about 10-20 h with HPLC after which performance gradually degraded as the AgCl on the reference electrode dissolved causing loss of potential control.     

Probing the binding behavior and conformational states of globular proteins in reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic solvent gradient elution conditions, typically carried out at ambient temperatures. These conditions can however induce conformational effects with proteins as evident from changes in their biological or immunological activities. By monitoring the influence of temperature on the retention and band-broadening characteristics of proteins, the role of conformational processes in these lipophilic environments can be examined. These processes can then be interpreted in terms of a two-state model involving a native (N) and a fully unfolded species (U) or more complex folding/unfolding models. In the present study, the gradient elution RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temperatures between 5 and 85 degrees C with n-octadecyl (C18)- and n-butyl (C4)-silica reversed-phase sorbents. The interaction of these proteins with these reversed-phase sorbents has also been examined in terms of the contributions that the heme prosthetic group of SWMYO and the disulfide bonds in HEWL make to the stabilization of the native conformation of these proteins in these hydrophobic environments. The observed interconversions of multiple peak zones of SWMYO and HEWL in the presence of C18 and C4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conformational intermediates of proteins in these perturbing environments has been examined. Pseudo-first-order rate constants have been derived for these processes from analysis of the dependencies on time of the concentration of the different protein species at specified temperatures. The relationship of these processes to the conformational transitions that these proteins can undergo via molten globule-like intermediates (i.e., compact denatured states with a significant amount of residual secondary structure) in solution has also been examined. This study thus further documents an experimental strategy to assess the folding/unfolding behavior of globular proteins in the presence of hydrophobic surfaces and aquo-organic solvents, whereby the system parameters can potentially affect the preservation of native conformations, and thus the function, of the protein under these conditions.     

Analysis of oligosaccharides by microbore high-performance liquid chromatography

A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.     

Nanoflow gradient generator for capillary high-performance liquid chromatography

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.     

Validation of tablet dissolution method by high-performance liquid chromatography

Dissolution is a qualitative and quantitative tool that can provide valuable information about biological availability of a drug, as well as batch-to-batch consistency. It is considered one of the most important quality control tests performed on pharmaceutical dosage forms, and validation of dissolution methods is an important part of good manufacturing practices (GMP). Hydroxypropylcellulose (HPC) was formulated with acetaminophen (APAP) and hydrochlorothiazide (HCTZ). Dissolution methods and limits are reported in the USP/NF. Standard operating procedures (SOP) for the HP 8452A spectrophotometer and Vanderkamp 600/6010 Dissolution Tester were followed according to the GMP Manual. A dissolution method was developed for each formulation based on the above. The only discrepancy between high-performance liquid chromatography (HPLC) and standard dissolution testing occurred when comparing the results of the HPC/HCTZ formulation. The ultraviolet (UV) samples were filtered through a 10-μm filter, and the HPLC samples were filtered through a 0.2-μm filter. When the HPC/HCTZ samples were filtered through a 10-μm filter for both UV and liquid chromatography (LC), the results were equal. Filter pore size and area have a large effect on concentration of HPC/HCTZ. The smaller the pore size and the smaller the diameter of the filter, the more HPC/HCTZ is filtered out. HCTZ has a greater tendency to interact with HPC in the filter than other active ingredients tested. HPC and HCTZ levels have little or no effect on the amount of HCTZ lost     

Determination of lipophilicity by gradient elution high-performance liquid chromatography

A novel method for the determination of lipophilicity using a simple HPLC protocol based on gradient elution chromatography is presented and compared to the common isocratic log k'(w) procedure. Linear relationships with high correlation coefficients between both methods for biologically active nucleosides and cyclic nucleotides as well as for environmentally relevant aromatic hydrocarbons were found. A mathematical fit to support the empirically determined linear relationship is presented. It is shown that the observed relationship between log k'(w) and the apparent capacity factor (k'(g)) determined by gradient elution is derivable by theoretical considerations as well. Since the gradient method is much less time-consuming compared to other procedures, it represents a convenient alternative for determining lipophilicity data in the future.     

Vancomycin dimerization and chiral recognition studied by high-performance liquid chromatography

The retention and separation of D,L-dansylvaline enantiomers (used as test solutes) were investigated using silica gel as stationary phase and vancomycin as chiral mobile-phase additive. A retention model was developed to describe the mechanistic aspects of the interaction between solute and vancomycin in the chromatographic system. It considered the formation of vancomycin dimers both "free" in the mobile phase and adsorbed on silica. By fitting the model equation to experimental data, it appeared clearly that the approach taking into account the vancomycin dimerization described accurately the retention behavior of the compounds. The examination of the model equation parameters showed that the glycopeptide dimerization increased the enantioselectivity by a factor of approximately 3.7. This study demonstrated the preponderant role of the vancomycin dimerization on the chiral recognition process of D,L-dansylvaline. Also, an additional analysis on a vancomycin chiral stationary phase indicated that the addition of vancomycin in the mobile phase promoted a greater enantioselectivity mediated by the formation of dimers in the stationary phase.     

An improved high-performance liquid chromatography assay for spironolactone analysis

This study prepared an extemporaneously formulated liquid suspension dosage form (5 mg/ml) from commercially available 25 mg tablets. Stability-indicating HPLC assay procedures were established and utilized to analyze the concentration of the drug. The method proved to be a simple model since it does not contain a buffer system. The mobile phase used was the same as that suggested by the manufacturer for the storage of the column. Therefore, the solvent system saves analytical processing time, since it does not require a change in the mobile phase before or after the analysis. The analytical method has been shown to be stability-indicating. The results have shown that there is no interference from any of the degradation products obtained from stressing spironolactone by heat and extremes in pH or with the internal standard, hydrocortisone 21-acetate.     

A high-performance liquid chromatography assay for yohimbine HCl analysis

The analysis used yohimbine HCl solution prepared from commercially available yohimbine HCl powder. Stability-indicating high-performance liquid chromatographic (HPLC) assay procedures were established and utilized to analyze the concentration of the drug. The method proved to be a simple model since it does not contain a buffer system. The mobile phase used, a methanol:water 70:30 ratio, was similar to that suggested by the manufacturer for the storage of the column. Therefore, the solvent system saves analytical processing time since it does not require a change in the mobile phase before and after the analysis. The analytical method has been shown to be stability indicating. The assay method showed a retention time for yohimbine of 4.2 min; for caffeine, the internal standard, it was 2.3 min. The standard deviation and the coefficient of variation were under acceptable limits of 2% and were specifically 1.51% and 1.35% for within-day and between-day samples, respectively. The results showed that the degradation products obtained from stressing yohimbine HCl by heat and extremes in pH did not interfere with the yohimbine HCl peak, although the internal standard, caffeine, did show some interference due to having a retention time similar to the degradation products.     

DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.     

Separation of nonionic surfactants by capillary electrophoresis and high-performance liquid chromatography

Fatty alcohol ethoxylates (FAEs) are applied in commercial formulations (laundry detergents) as complex mixtures of alkyl and ethoxylate homologues. Therefore, efficient analytical methods are required for product control. Capillary electrophoresis, a modern analytical separation technique, was used to separate FAEs in technical products and household formulations after derivatization with phthalic anhydride. The well-established high-performance liquid chromatography was used as reference and supplementary method. UV detection after derivatization with phenyl isocyanate or light scattering detection has been carried out. Sample components have been identified by standard addition or by comparison to known products. The peak pattern can be considered as a "fingerprint" of the product and is characteristic for a defined composition.     

Selective and quantitative detection of influenza virus proteins in commercial vaccines using two-dimensional high-performance liquid chromatography and fluorescence detection

In this work, we report on the applicability of two-dimensional high-performance liquid chromatography (2D-HPLC) for the comprehensive characterization of inactivated influenza vaccine proteins. This novel procedure features minimal sample treatment and combines the on-line coupling of size exclusion HPLC to reversed-phase HPLC. A comparative analysis of commercial vaccines from three different manufacturers showed the method to be highly selective by providing characteristic reproducible chromatographic profiles for each vaccine. In addition, the method provided enhanced sensitivity for most constituents as a result of the use of native fluorescence detection in the reversed-phase HPLC step. The limits of detection (at a signal-to-noise ratio of >3) for hemagglutinin (HA) antigens were 105 and 172 ng/mL for influenza A/New Caledonia/20/99 and B/Jiangsu/10/2003 strains, respectively. The potential of this 2D-HPLC procedure in terms of quantitative antigen analysis was assessed by determination of the HA content of commercial vaccines. Results provided very good correlation with nominal HA values. The reproducibility (RSD) of the whole procedure was also evaluated and was found to be better than 2 and 3% for calculated antigen concentrations expressed as micrograms of HA per milliliter in commercial vaccines for samples of the same lot (n = 5) or different lots (n = 3), respectively. In addition, it allowed the selective detection of several influenza constituents including nucleoproteins from type A and B viruses and the highly hydrophobic matrix protein 1 from both virus strains.     

Overload for ionized solutes in reversed-phase high-performance liquid chromatography

Overloading occurs for submicrogram quantities of ionized solutes particularly when using low ionic strength mobile phases at low pH (e.g., formic acid), even with highly inert silica RP-HPLC columns of normal dimensions. Much higher loads can produce a sharp L-shaped peak with retention above the column void volume, in line with the hypothesis that a small number of high-energy sites fill first and are rapidly overloaded, followed by a much larger number of weaker sites. However, charged acids and bases show identical overloading behavior; overloading is reduced as the mobile-phase ionic strength is increased. These findings raise questions about the physical nature of the strong sites. The rapid overloading of silica and purely polymeric phases could be explained by mutual repulsion of ionic species or their inability to fully penetrate the hydrophobic structure of the phase. However, these alternative hypotheses cannot readily explain the high total saturation capacities obtained using frontal analysis. Ion pairing with trifluoroacetic acid may reduce overload, while the effect is less important for formate or phosphate buffers. A surface layer of acetonitrile is not a prerequisite for rapid overloading, as shown by studies using purely aqueous buffers.     

Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A

Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.