On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.     

Array based capillary IEF with a whole column image of laser-induced fluorescence in coupling to capillary RPLC as a comprehensive 2-D separation system for proteome analysis

Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.     

Fibrous stationary phase in capillary electrochromatography

Capillary electrochromatography (CEC) using fibrous cellulose acetate (CA) stationary phase was investigated. The advantage of this fiber-packed column is relatively easy preparation process compared with other conventional CEC columns, such as particle-packed and wall-coated capillaries. CA fibers are manually packed into a capillary with two guide liners and fixed with a frit at the column inlet. The separation characteristics of this column were investigated using n-alkyl p-hydroxybenzoates (parabens) as the sample probe. It has been demonstrated that the use of a short column length and a specially designed tee-connector as the injection device should make the separation performance and efficiency much higher on the fiber-packed columns. Sufficient separation between methyl and n-butylparabens is obtained on the 5-cm-packed column and linear relationships between the injection time and the peak area are observed. Bubble formation is not encountered during the analysis.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Electrochromatographic characterization of methacrylate ester-based monolith and capillary electrochromatography separation of flavonoids

A porous polymethacrylate ester-based monolithic column for capillary electrochromatography (CEC) was designed by mean of in situ co-polymerizing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent including cyclohexanol, 1,4-butanediol and water. After investigating the influence factors of the CEC monolithic columns, four flavonoids (i.e., Rutin, Quercetin, Kaempferol, and Quercitrin) were separated and assayed to evaluate this monolithic column with CEC method. Under optimum conditions, the CEC method exhibited high separation efficiency, with rapid separation time of 3–4 min, for the four flavonoid samples using 10 mM phosphate buffer containing 70% acetonitrile (pH 9.0). Importantly, the proposed method could provide a promising approach for rapid separation and detection in biomedicine.     

Integrated capillary isoelectric focusing/nano-reversed phase liquid chromatography coupled with ESI-MS for characterization of intact yeast proteins

An integrated protein concentration/separation platform, combining capillary isoelectric focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction "parked" in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry.     

Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies

Capillary isoelectric focusing (CIEF) involves the use of the entire capillary filled with a mixture containing protein/peptide analytes and carrier ampholytes. Thus, the preparative capabilities of CIEF are inherently greater than most capillary-based electrokinetic separation techniques. To further increase sample loading and, therefore, the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from a solution reservoir, is demonstrated using a low p/ protein calibration kit and tryptic peptides from Saccharomyces cerevisiae. The proteins/peptides continuously migrate into the capillary and encounter a pH gradient established by carrier ampholytes originally present in the capillary for focusing and separation. Dynamic introduction and focusing in CIEF can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Depending on the mobilities of yeast peptides, the loading capacity of each peptide is measured to be around 8 to 45-fold of that obtained in conventional CIEF. By comparing with the concentrations of dilute yeast peptides originally present in the reservoir, an overall concentration factor of 1400-7700 together with excellent separation resolution is achieved using dynamic introduction and focusing. This concentration effect is further illustrated by detecting 10 pg/microL of bradykinin peptide spiked in yeast protein digest using only ultraviolet absorption.     

Heart-cut two-dimensional separation method via hyphenation of micellar electrokinetic capillary chromatography and capillary zone electrophoresis using analyte focusing by micelle collapse

A novel two-dimensional (2D) separation method, which hyphenated micellar electrokinetic capillary chromatography (MEKC) and capillary zone electrophoresis (CZE), was developed for analysis of flavonoids in Leonurus cardiaca. The Leonurus cardiaca sample was separated and purified in first dimension by MEKC. Then only a selected portion of the first dimension separation was transferred into the second dimension by pressure. Finally, the zone of flavonoids was separated by CZE. As the key to successful hyphenation of MEKC and CZE, an analyte focusing by micelle collapse (AFMC) concentration method was employed between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The whole heart-cut 2D separation process can be performed in a conventional CE analyzer. The relative standard deviation of peak height, peak area and migration time were in the range of 2.3-4.2%, 1.5-3.8% and 3.6-5.5%, respectively, and detection limits (S/N=3) were 15-55 ng/mL. The new methodology was applied with success for the flavonoids separation of Leonurus cardiaca.     

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.     

Analysis of human blood serum using the off-line coupling of capillary isoelectric focusing to matrix-assisted laser desorption/ionization time of flight mass spectrometry

Off-line coupling of capillary IEF (CIEF) with matrix-assisted laser desorption/ionization mass spectrometry was utilized for the analysis of human blood serum. Serum proteins were initially separated by CIEF, and fractions of the isoelectric separation were eluted sequentially to a MALDI-TOF MS sample target. During pressure elution of the CIEF sample, voltage was maintained across the capillary system utilizing a sheath flow arrangement to minimize band broadening induced by the laminar flow field. Both pI and mass information were obtained from the complex biological sample, similar to traditional 2-DE techniques, and the platform was faster (hours versus days), more automatable, and simpler than 2-DE. The volume of raw sample present in the actual analysis was approximately 100 nL, making this technique well suited for very rare specimens. Additionally, the speed and simplicity of the technology make it an attractive technique for performing initial comparative analyses of complex samples.     

Separation of microcystins by capillary electrochromatography in monolithic columns

Contribution on microcystin variant analysis by capillary electrochromatography (CEC) with easily affordable spectrophotometric detection is presented. Two types of reversed-phase capillary columns formed by inorganic or organic polymer monoliths were prepared for this purpose. The analyses were performed isocratically by means of tris(hydroxymethyl) aminomethane (TRIS) buffers of mildly alkaline pH containing 30% (v/v) acetonitrile as the mobile phases. The samples were injected electrokinetically and the analyses were done at the same separation field strength of 500 V/cm. Microcystins were detected at 238 nm. Although both column types differ not only in monolith quality (inorganic versus organic) but also in the length of the aliphatic moiety (C8 versus C12) similar results were achieved. The on-column preconcentration as the encouraging prospect of electrochromatographic technique was also tested. Consequently 5% of column volume was injected in contrast with 0.5% at standard injection scheme resulting in the six times enrichment of the low concentrated cyanobacterial extract at the top of the separation column. From these preliminary results can be seen that the CEC method is fully applicable for rapid microcystin screening.     

Enhanced Sequence Coverage in Tryptic Fragment Analysis by Two-Dimensional HPLC/MS Using a Monolithic Silica Capillary Column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

The covalently bonded cellulose tris(3,5-dimethylphenylcarbamate) on a silica monolithic capillary column for enantioseparation in capillary electrochromatography

A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-microm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.     

Development of a highly efficient 2-D system with a serially coupled long column and its application in identification of rat brain integral membrane proteins with ionic liquids-assisted solubilization and digestion

Two dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-HPLC-ESI-MS/MS) is one of the most powerful techniques for high resolution, efficiency, and throughput separation and identification of proteomes. For a bottom-up strategy-based proteome analysis, usually multistep salt elution was needed in the first dimension separation by SCX, to simplify the peptides for the further second dimensional separation by RPLC. Here, by using a 30 cm-long serially coupled long column (SCLC) in the second dimension, we reduced the salt steps of SCX from 13 to 5 to shorten the total analysis time. Compared to the commonly applied 2D-HPLC with over 10-step salt elution in SCX and microRPLC with a short column (SC), named as SC-2D, the peak capacity of 2D-HPLC with a SCLC column, named as SCLC-2D, was increased 3.3-folds while the analysis time was increased by only 1.17-folds. Therefore, the time-based protein identification efficiency was ～55 protein groups/h, nearly 2-fold of that for SC-2D (～28 protein groups/h). With the further combination of assisted solubilization by ionic liquids and SCLC-2D, 608 integral membrane proteins (IMPs) (27.66% of the total 2198 proteins, FDR < 1%) were identified from rat brain, more than those obtained by the traditional urea method (252 unique IMPs, occupying 17.03% of total 1480 proteins). All of these results demonstrate the promise of the developed technique for large-scale proteome analysis.     

Octadecylated silica monolith capillary column with integrated nanoelectrospray ionization emitter for highly efficient proteome analysis

An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 mug of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution.     

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.     

Automated glass capillary gas-liquid chromatography of fatty acid methyl esters with reference to cis and trans isomers.

The availability of an excellent separation method for fatty acid methyl esters, including separation of cis and trans isomers and of isomers that differ only in the position of double bonds, has become more and more important. The present glass capillary chromatography system combines high separation power with high precision and easy handling. Moreover, the system is completely automated and therefore provides a time saving method. As compared to a conventional packed column, the glass capillary column provides about one hundred fold more theoretical plates (227,000), as well as narrower peaks, thus giving rise to less error when integrating with electronic integrators. The reproducibility for relative retention time is better with the capillary column (0.26%) and reproducibility of the weight percent values is at least similar to that of the packed column (1.53%). When handling only small sample amounts the capillary provides better values because of its low capacity. This powerful system should open up new possibilities in the field of fatty acid investigation.     

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.     

Experimental design as a tool when evaluating stationary phases for the capillary electrochromatographic separation of basic peptides

Two different capillary electrochromatography (CEC) stationary phases, Hypersil phenyl and Hypersil C(18), have been characterised with respect to their ability to separate the four basic peptides H-Tyr-(D)Ala-Phe-Phe-NH(2) (TAPP), H-Tyr-(D)Ala-Phe-NH(2) (TAP), H-Phe-Phe-NH(2) (PP) and H-Phe-NH(2) (P). Optimal separation conditions were first established separately for the two phases by applying experimental design in a stepwise procedure. The first step comprised a study to acquire basic knowledge about the variables, their influence on the response and their respective experimental domains for each of the two stationary phases. The second step was screening the significant variables and the third step was an optimisation with response surface modelling (RSM) to locate the optimum separation conditions for each stationary phase. The experimental procedure was identical for both stationary phases, but their respective experimental domains were different. The response functions were peak resolution and peak efficiency. This procedure enables specific optimal experimental conditions to be identified for each of the two stationary phases. The optimal conditions identified for the separation on the phenyl stationary phase were to use 50% ACN, 20% 50 mM Tris(hydroxymethyl)aminomethane (TRIS) pH 7.5, 30% H(2)O as BGE, operating at 20 degrees C and 20 kV high voltage. For the C(18) stationary phase optimal separation was achieved using a BGE with 80% ACN, 20% 30 mM TRIS pH 8.5, again operating at 20 degrees C and 20 kV high voltage. Results show that the phenyl stationary phase is better suited for the separation of basic, hydrophilic peptides.