A multidimensional differential proteomic platform using dual-phase ion-exchange chromatography-polyacrylamide gel electrophoresis/reversed-phase liquid chromatography tandem mass spectrometry

Differential proteomic analysis has arisen as a large-scale means to discern proteome-wide changes upon treatment, injury, or disease. Tandem protein separation methods are required for large-scale differential proteomic analysis. Here, a novel multidimensional platform for resolving and differentially analyzing complex biological samples is presented. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines biphasic ion-exchange chromatography with polyacrylamide gel electrophoresis for protein separation, quantification, and differential band targeting, followed by capillary reversed-phase liquid chromatography and data-dependent tandem mass spectrometry for quantitative and qualitative peptide analysis. CAX-PAGE provides high protein resolving power with a theoretical peak capacity of 3570, extendable to 7600, a wide protein mass range verified from 16 to 273 kDa, and reproducible differential sample comparison without the added expense of fluorescent dyes and imaging equipment. Demonstrated using a neuroproteomic model, CAX-PAGE revealed an increased number of differential proteins, 137, compared with 82 found by 2D difference gel electrophoresis. When combined with RPLC-MSMS for protein identification, an additional quantification step is performed for internal validation, confirming a 2-fold or greater change in 89% of identified differential targets.     

Thermodynamic and kinetic characterization of polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography

The retention of six polycyclic aromatic hydrocarbons (PAHs) was characterized by reversed-phase liquid chromatography. The PAHs were detected by laser-induced fluorescence at four points along an optically transparent capillary column. The profiles were characterized in space and time using an exponentially modified Gaussian equation. The resulting parameters were used to calculate the retention factors, as well as the concomitant changes in molar enthalpy and molar volume, for each PAH on monomeric (2.7 micromol/m2) and polymeric (5.4 micromol/m2) octadecylsilica. The changes in molar enthalpy become more exothermic as ring number increases and as annelation structure becomes less condensed. The changes in molar volume become more negative as ring number increases for the planar PAHs, but are positive for the nonplanar solutes. In addition, the rate constants, as well as the concomitant activation enthalpy and activation volume, are calculated for the first time. The kinetic data demonstrate that many of the PAHs exhibit very fast transitions between the mobile and stationary phases. The transition state is very high in energy, and the activation enthalpies and volumes become greater as ring number increases and as annelation structure becomes less condensed. The changes in thermodynamic and kinetic behavior are much more pronounced for the polymeric phase than for the monomeric phase.     

Extrathermodynamic relationships in reversed-phase liquid chromatography

Enthalpy-entropy compensation (EEC) and linear free energy relationships (LFER) are extrathermodynamic correlations frequently used to discuss the mechanistic similarities of chemical equilibria and reaction kinetics. Although empirical, they are widely applied, proving the substantial effectiveness of fundamental studies based on them. Many attempts have been made to interpret theoretically the necessary conditions (or preconditions) of EEC and LFER. LFER is known to rest on the existence of EEC. However, the intimate correlations between EEC on one hand and LFER and the temperature dependence of LFER on the other hand were insufficiently discussed from the viewpoint of molecular structure contributions. We present a simple LFER model relating the slope and intercept of LFER to the compensation temperatures, themselves derived from EEC analyses, and to several parameters characterizing the molecular contributions to the changes in enthalpy and entropy associated with the passage from one phase of the chromatographic system to the other. A theoretical explanation is supplied for the intimate correlation between the two types of extrathermodynamic relationships, EEC and LFER. We demonstrate also that the characteristics of EEC and LFER depend on the structural parameters. This new model allows a proper interpretation of the temperature dependence of LFER. It should permit further progress of fundamental studies of chemical reaction mechanisms based on extrathermodynamic relationships.     

Moment analysis of mass-transfer kinetics in C18-silica monolithic columns

The moment analysis of elution peak profiles based on new moment equations provides information on the mass-transfer characteristics of C(18)-silica monolithic columns. The flow rate dependence of the HETP data was analyzed using the generalized van Deemter equation, after correction of these data by subtraction of the external mass-transfer contribution to band broadening. Kinetic parameters and diffusion coefficients related to the mass-transfer processes in monolithic columns were derived by taking advantage of the different flow velocity dependence of their contributions to band broadening. At high flow rates, axial dispersion and diffusive migration across the monolithic C(18)-silica skeleton contribute much to band broadening, suggesting that it remains important to reduce the influence of eddy diffusion and the mass-transfer resistance in the stationary phase to achieve fast separations and a high efficiency. Surface diffusion plays a predominant role for molecular migration in the monolithic stationary phase. Although the value of the surface diffusion coefficient (D(s)) depends on an estimate of the external mass-transfer coefficient, D(s) values of the order of 10(-7) cm(2) s(-1) were calculated for the first time for the C(18)-silica monolithic skeleton. The value of D(s) decreases with increasing retention of sample compounds. Analysis of a kind of time constant calculated from D(s) suggests that the "chromatographic corresponding particle size" is approximately 4 microm for the C(18)-silica monolithic stationary phase used in this study. The accuracy of the D(s) values determined was discussed.     

Electrogenerated chemiluminescence detection in reversed-phase liquid chromatography

Electrogenerated chemiluminescence (ECL) has been developed as a detection method for liquid chromatography. The radical cation of tri-p-tolylamine (TPTA) is used as a common electron acceptor for the electrogenerated radical anions of a variety of organic analytes. ECL is accomplished with a high-frequency potential pulse program applied to a microelectrode immersed in the column eluent. ECL detection is demonstrated with reversed-phase liquid chromatography. Selectivity at the ECL detector is shown to be tunable based on differing electrochemical conditions and excited-state energetics. Low minimum detection limits in ECL are attributed to the dependence on the photon detector shot noise, allowing a limit of detection of 0.14 nM for perylene in the presence of 0.1 mM TPTA. A derivatization agent useful for ECL detection is demonstrated by the use of naphthalene-2,3-dicarboxaldehyde. This reagent, which does not itself result in ECL, forms ECL candidates following reaction with primary amines.     

Retention mechanism in reversed-phase liquid chromatography: a molecular perspective

A detailed, molecular-level understanding of the retention mechanism in reversed-phase liquid chromatography (RPLC) has eluded analytical chemists for decades. Through validated, particle-based Monte Carlo simulations of a model RPLC system consisting of dimethyloctadecylsilanes at a coverage of 2.9 micro mol/m2 on an explicit silica substrate with unprotected residual silanols in contact with a water/methanol mobile phase, we show that the molecular-level retention processes for nonpolar and polar analytes, such as alkanes and alcohols, are much more complex than what has been previously deduced from thermodynamic and theoretical arguments. In contrast to some previous assumptions, the simulations indicate that both partitioning and adsorption play a key role in the separation process and that the stationary phase in RPLC behaves substantially different from a bulk hydrocarbon phase. The retention of nonpolar methylene segments is dominated by lipophilic interactions with the retentive phase, while solvophilic interactions are more important for the retention of the polar hydroxyl group.     

Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics

Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.     

A study of the enthalpy and entropy contributions of the stationary phase in reversed-phase liquid chromatography

The goal of this study was to elucidate the roles played by the stationary and mobile phases in retention in reversed-phase liquid chromatography (RPLC) in terms of their individual enthalpic and entropic contribution to the Gibbs free energy of retention. The experimental approach involved measuring standard enthalpies of transfer of alkylbenzenes from typical mobile phases used in RPLC (methanol/water and acetonitrile/water mixtures), as well as from n-hexadecane (a simple analogue of the stationary phase) to the gas phase, using high-precision headspace gas chromatography. By combining the measured enthalpies with independently measured free energies of transfer, the entropies of transfer were obtained. This allowed us to examine more fully the contribution that each phase makes to the overall retention. It was found that the standard enthalpy of retention in RPLC (i.e., solute transfer from the mobile phase to the stationary phase) is favorable, due to the large and favorable stationary-phase contribution, which actually overcomes an unfavorable mobile-phase contribution to the enthalpy of retention. Further, the net free energy of retention is favorable due to the favorable enthalpic contribution to retention, which arises from the net interactions in the stationary phase. Entropic contributions to retention are not controlling. Therefore, to a great extent, retention is due to enthalpically dominated lipophilic interaction of nonpolar solutes with the stationary phase and not from solvophobic processes in the mobile phase. Further, our enthalpy data support a "partition-like" mechanism of retention rather than an "adsorption-like" mechanism. These results indicate that the stationary phase plays a very significant role in the overall retention process. Our conclusions are in direct contrast to the solvophobic model that has been used extensively to interpret retention in RPLC.     

Probing strong adsorption of solute onto C18-silica gel by fluorescence correlation imaging and single-molecule spectroscopy under RPLC conditions

Understanding molecular adsorption at a chromatographic interface is of great interest for addressing the tailing problem in chemical separations. Single-molecule spectroscopy and confocal fluorescence correlation imaging are used to study the adsorption sites of C(18) silica beads under RPLC chromatographic conditions. The experiments show that cationic molecule rhodamine 6G laterally diffuses through the chromatographic interface of a C(18) hydrocarbon monolayer and acetonitrile with occasional reversible strong adsorptions. Fluorescence correlation imaging extracts the rare strong adsorption events from large data sets, revealing that the strong adsorption sites are randomly distributed throughout the silica beads. Virtually every imaging pixel of silica beads adsorbs molecules. Single-molecule spectroscopy of the 584 strong adsorption events observed indicates that the strong adsorptions persist on the time scales from several milliseconds to seconds, having an average desorption time of 61 ms. The strong adsorption events are rare, comprising 0.3% of the total observation time. The sizes of strong adsorption sites are within the optical resolution of confocal imaging.     

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.     

Shape selectivity for constrained solutes in reversed-phase liquid chromatography

In reversed-phase liquid chromatography (RPLC), the separation of compound mixtures of similar polarity can present a significant challenge for the analyst. Examples of such compounds include geometric isomers present in environmental samples (e.g., polycyclic aromatic hydrocarbons, polycyclic aromatic sulfur heterocycles, and polychlorinated biphenyl congeners) and compounds of biological significance (e.g., carotenoids and steroids). In general, compounds with rigid, well-defined molecular shape are best separated using a column with enhanced shape selectivity characteristics. This perspective presents an overview of column properties that influence shape selectivity for constrained solutes. Approaches to the characterization of stationary-phase structure are described, and the findings are correlated with chromatographic performance. Finally, retention models of shape discrimination are presented that are consistent with observed retention behavior. An appreciation for shape recognition effects in RPLC will facilitate method development for certain classes of difficult to resolve compounds.     

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.     

Characterization of currently marketed heparin products: reversed-phase ion-pairing liquid chromatography mass spectrometry of heparin digests

Here we report results from the analyses by enzymatic digestion and reversed-phase ion-pairing liquid chromatography mass spectrometry (RPIP-LC-MS) of active pharmaceutical ingredient (API) unfractionated heparins (UFHs) from six different manufacturers and one USP standard sample. We employed a reverse phase ion-pairing chromatography method using a C(18) column and hexylamine as the ion-pairing reagent with acetonitrile gradient elution to separate disaccharides generated from the digestion of the heparins by lyase I and III (E.C. 4.2.2.7 and 4.2.2.8) before introduction into an ion-trap mass spectrometer by an electrospray ionization (ESI) interface. Extracted ion chromatograms (EICs) were used to determine the relative abundance of the disaccharides by mass spectrometry. Eight disaccharides were observed and a similar composition profile was observed from digests of 20 UFH samples. The compositional profile determined from these experiments provides a measure of the norm and range of variation in "good" heparin to which future preparations can be compared. Furthermore, the profile obtained in the RPIP-LC-MS assay is sensitive to the presence of the contaminant, oversulfated chondroitin sulfate A (OSCS), in heparin.     

Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ～6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.     

Elution of propranolol as an ion-pair complex by buffer solutions on C18-silica

Propranolol (pK(a) = 9.4) was eluted on C(18)-bonded Kromasil, equilibrated with buffer solutions of methanol and water (40/60, v/v) containing a constant concentration of a counteranion (12 mM). Nine different counteranions were studied: Cl(-), I(-), NO(3)(-), SO(4)(2-), CH(3)COO(-), HOOCC(2)H(4)COO(-), (-)OOCC(2)H(4)COO(-), HOOCCOHCOOHCOO(-), HOOCCOHCOO(-)COO(-), and (-)OOCCOHCOO(-)COO(-). The co-cation was K(+) or Na(+). Vacancy perturbations were measured on three concentration plateaus of propranolol hydrochloride, at 1.2, 12, and 24 mM, by injecting 100 microL of a pure mixture of methanol and water (40/60, v/v). Indirect detection of the solvent, the counteranion, the co-cation, and the chloride ion was carried out at 325 nm, a wavelength at which only propranolol responds. In a 1.2 mM propranolol hydrochloride solution, there is a 10-fold excess of counteranions and only a positive perturbation peak, due to the excluded co-cation and eluting before the column hold-up time, and a large vacancy peak, associated with propranolol, were recorded. Association between propranolol and the counteranion in excess determines the retention time of this second perturbation. The hydrophobicity of the complexes increases in the order Cl(-) < CH(3)COO(-) approximately HOOCC(2)H(4)COO(-) < NO(3)(-) < I(-) < HOOCCOHCOOHCOO(-) < (-)OOCC(2)H(4)COO(-) < SO(4)(2-) approximately HOOCCOHCOO(-)COO(-) < (-)OOCCOHCOO(-)COO(-). Propranolol retention is larger in the presence of the trivalent citrate anion than in that of the bivalent citrate, succinate, or sulfate anions. It is larger with these bivalent anions than with any monovalent anion. Equal concentration of propranolol hydrochloride and buffer in the mobile phase reveals five system peaks associated with the five components (solvent, counteranion, co-cation, chloride, propranolol molecules). In contrast with monovalent anions, bivalent anions (sulfate, succinate, citrate) or trivalent anions (citrate) cause a reversal of the elution order of the perturbation peaks of chloride anions and buffer molecules. This confirms a competition between chloride and buffer anions to form ion pairs with propranolol. The retention of the perturbation signal of the buffer increases with increasing anion charge because multivalent anions can bind to several molecules of propranolol. The perturbation measurements demonstrate the influence of the valence and hydrophobicity of the buffer on the retention of ionizable compounds. The inverse method allowed the derivation of the isotherm parameters from the overloaded band profiles of propranolol. These values confirm that adsorbate-adsorbate interactions increase with increasing valence of the anions.     

Determination of fluorescence-labeled asparaginyl-oligosaccharide in glycoprotein by reversed-phase ultraperformance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.