Coupling of solid-phase microextraction and capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection for protein analysis

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEF-WCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, R-phycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 x 10(-12) M (S/N = 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells.     

Fluorescence-labeled peptide pI markers for capillary isoelectric focusing

Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection. Tetra- to tridecapeptides containing one cysteine residue were designed to focus sharply at their respective isoelectric points by including amino acids that contain charged side chains, the pKa values of which are close to the corresponding pI values. An iodoacetylated derivative of tetramethylrhodamine was coupled to the thiol group of cysteine to yield fluorescent pI markers. The pI values of the labeled peptides were precisely determined after isoelectric focusing on polyacrylamide gel slabs by direct measurement of the pH of the focused bands. The markers were subjected to capillary isoelectric focusing for 10-15 min in coated capillaries under conditions of low electroosmosis and were detected by means of a scanning laser-induced fluorescence detector down to a level of subpicomolar range. The markers permitted the calibration of a wide-range pH gradient formed in a capillary by fluorescence detection for the first time and should facilitate the development of highly sensitive analytical methods based on a combination of capillary isoelectric focusing and laser-induced fluorescence detection.     

Accuracy in the determination of isoelectric points of some proteins and a peptide by capillary isoelectric focusing: utility of synthetic peptides as isoelectric point markers

To evaluate the accuracy ofisoelectric point determination by capillary isoelectric focusing, the pI values of nine proteins and a peptide, the pI values of which had been determined by other methods and ranging pI 3.55-9.60, were determined by capillary isoelectric focusing by cofocusing of recently developed peptide pI markers ranging 3.38-10.17, and the consistency of the pI values was examined. Isoelectric focusing was carried out in neutral polymer-coated capillaries, and the pH gradient was mobilized by pressure toward the cathode, to detect samples with absorption at 280 nm at a fixed detection point. Carrier ampholytes from two different suppliers and in different pH ranges were used. The sharp peaks of the highly pure peptide pI markers greatly facilitated the unambiguous identification of the peaks. When a carrier ampholyte ranging over the acidic side was used, the detection of acidic pI samples was anomalously delayed. This could be partly mitigated by reducing the viscosity of the anode solution in comparison with the pH gradient formed in the capillary. Since the detection times vs the pH relationships were not linear in most cases, the use of a linear calibration line over an entire pH gradient would be erroneous. Instead, the pI values of samples were calculated by assuming a linear relation for pH against detection time between two flanking marker peptides. Close agreement between the pI values, determined by capillary isoelectric focusing, and the reference values of the samples was observed within an average difference range of 0.04-0.08 pH unit with a sample consumption of 10-100 ng within 30-60 min. Some carrier ampholytes were preferentially more effective at either the acidic or the basic side of the pH gradient. For confirmation of the completion of focusing, the use of two different focusing times is recommended.     

Capillary isoelectric focusing and fluorometric detection of proteins and microorganisms dynamically modified by poly(ethylene glycol) pyrenebutanoate

The nonionogenic pyrene-based tenside, poly(ethylene glycol) pyrenebutanoate, was prepared and applied in capillary isoelectric focusing with fluorometric detection. This dye was used here as a buffer additive in capillary isoelectric focusing for a dynamic modification of the sample of proteins and microorganisms. The values of the isoelectric points of the labeled bioanalytes were calculated with use of the fluorescent pI markers and were found comparable with pI of the native compounds. The mixed cultures of proteins and microorganisms, Escherichia coli CCM 3954, Staphylococcus epidermidis CCM 4418, Proteus vulgaris, Enterococcus faecalis CCM 4224, and Stenotrophomonas maltophilia, the strains of the yeast cells, Candida albicans CCM 8180, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae were reproducibly focused and separated by the suggested technique. Using UV excitation for the on-column fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary.     

Nanoliter-volume selective sampling of peptides based on isoelectric points for MALDI-MS

A simple way to selectively isolate peptides based on their isoelectric points (pI) for MALDI mass spectral analysis is described. An applied voltage was used to electromigrate peptides into a capillary. The capillary was modified with a zwitterionic surfactant, 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), to suppress the electroosmotic flow (EOF) during injection. Hence, either the cationic or the anionic peptides were introduced, depending on the voltage polarity. By controlling the pH, selective loading of peptides was performed to isolate trace components from a mixture. The injected sample plugs were subsequently spotted in nanoliter volumes for MALDI-MS analysis. No significant sample losses resulting from selective sampling were detected. Low attomole-level detection of peptides (adrenocorticotropic hormone fragment 18-39, pI 4.25) was achieved from a mixture containing other peptides (angiotensin I, pI 6.92, and bradykinin, pI 12.00) at 100 000-fold higher concentrations.     

Isoelectric buffers for capillary electrophoresis. 2. Bismorpholine derivative of a carboxylic acid as a low molecular weight isoelectric buffer

A new compound class of synthetic isoelectric buffers is introduced, designed as a small molecule with one fully or prevailingly dissociated acidic group (such as sulfonic or carboxylic) and two partly pronated (buffering) basic amino groups attached onto a hydrophilic UV-transparent backbone. As an example, a new isoelectric compound 2,2-bis(4-morpholinylmethyl)propanoic acid (BMMPA) was synthesized by attaching two morpholine groups onto a molecule of pivalic acid. It was characterized as having an isoelectric point pI = 6.5 and exhibiting satisfactory buffering capacity at the pI. Solutions of BMMPA are transparent down to the low-UV spectral region, thus making it a potentially suitable buffer for a number of separation methods. Its use in capillary electrophoresis was demonstrated in a separation system for indirect photometric detection of anions based on an electrolyte with the anionic dye Orange G as the indirect detection probe and using BMMPA as a buffer. The use of an isoelectric buffering compound brings the advantages of a buffered electrolyte without the concomitant introduction of co-ions that would be detrimental to the indirect detection process. Submicromole per liter limits of detection for a number of inorganic and small organic ions were achieved. Optimal structural properties of the isoelectric buffer with respect to its buffering properties are discussed.     

Dynamics of single-protein molecules at a liquid/solid interface: implications in capillary electrophoresis and chromatography

The behavior of individual molecules of R-phycoerythrin (RPE) was monitored by fluorescence imaging at various pHs and ionic strengths within the evanescent-field layer (EFL) at a water/fused-silica interface. Above the isoelectric point (pI), the individual protein molecules moved between exposures with random motion. As the pH approached the pI of the protein, the RPE molecules were partially adsorbed onto the fused-silica surface. The residence time and the number of molecules within the EFL also increased near the pI. Below the pI, the protein molecules were completely and permanently adsorbed onto the surface. However, the observed number of distinct molecule spots was decreased somewhat because of aggregation. At a given buffer condition, plots of residence times and molecule numbers exhibit asymmetry nearly identical to the corresponding elution peaks of the proteins in capillary electrophoresis and capillary liquid chromatography. These results provide insights into the fundamental interactions for the adsorption/desorption of proteins at the liquid/solid interface.     

Capillary isoelectric focusing of individual mitochondria

Mitochondria are highly heterogeneous organelles that likely have unique isoelectric points (pI), which are related to their surface compositions and could be exploited in their purification and isolation. Previous methods to determine pI of mitochondria report an average pI. This article is the first report of the determination of the isoelectric points of individual mitochondria by capillary isoelectric focusing (cIEF). In this method, mitochondria labeled with the mitochondrial-specific probe 10-N-nonyl acridine orange (NAO) are injected into a fused-silica capillary in a solution of carrier ampholytes at physiological pH and osmolarity, where they are focused then chemically mobilized and detected by laser-induced fluorescence (LIF). Fluorescein-derived pI markers are used as internal standards to assign a pI value to each individually detected mitochondrial event, and a mitochondrial pI distribution is determined. This method provides reproducible distributions of individual mitochondrial pI, accurate determination of the pI of individual mitochondria by the use of internal standards, and resolution of 0.03 pH units between individual mitochondria. This method could also be applied to investigate or design separations of organelle subtypes (e.g., subsarcolemmal and interfibrillar skeletal muscle mitochondria) and to determine the pIs of other biological or nonbiological particles.     

Separation of plant pathogens from different hosts and tissues by capillary electromigration techniques

In this contribution capillary isoelectric focusing and capillary zone electrophoresis were applied for the separation and detection of different plant pathogens including Pseudomonas syringae pv. syringae, P. syringae pv. lachrymans, Pseudomonas savastanoi pv. fraxinus, P. savastanoi pv. olea, Agrobacterium tumefaciens, A vitis, Xanthomonas arboricola pv. juglandis, X. campestris pv. zinniae, and Curtobacterium sp.. The UV detection and sensitive fluorescence detection of the native phytopathogens or those dynamically modified by the nonionogenic fluorescent tenside based on pyrenebutanoate were used. The isoelectric points of the labeled phytopathogens were found comparable with the pI of the native compounds. No influence of the hosts on pIs of the strains of the genus Pseudomonas was observed. The identification of plant pathogens by gas chromatographic analysis of fatty acid methyl esters was compared with results of capillary isoelectric focusing. Capillary electromigration was successfully applied for the separation of microbes directly from plant tissue suspensions.     

High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms

High-resolution capillary isoelectric focusing separations of complex protein mixtures have been obtained for cellular lysates of Saccharomyces cerevisiae, Eschericia coli, and Deinococcus radiodurans. High quality separations are shown to be achievable for total protein concentrations of < 0.1 mg/mL. The separation reproducibility was examined, and the influence of the capillary inner wall coating on resolution investigated using fusedsilica capillaries coated with various hydrophilic polymers including hydroxypropyl cellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having an isoelectric point (pI) difference of 0.004 are shown to be separated using a linear carrier ampholyte (linear pH gradient between two electrodes) of 3-10. Approximately 45 discrete peaks in the pI range of 5-7 were obtained for S. cerevisiae, approximately 80 peaks in the pI range of 4.5-8.5 for E. coli, and approximately 210 peaks in the pI range of 3-8.8 for D. radiodurans.     

High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis

On-line capillary isoelectric focusing (CIEF)-electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of hemoglobin variants A, C, S, and F. The effects of moving ionic boundary inside the CIEF capillary and MS scan rate on the separation resolution and mass detection of hemoglobin variants are investigated. The formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. Hemoglobin variants F and A, with a pI difference of 0.05 pH unit, are almost baseline resolved and identified in CIEF-ESIMS. The concentration detection limit for each hemoglobin variant is in the range of 10(-)(8) M, comparable to that obtained in two-dimensional gel electrophoresis using silver staining. Initial preconcentration during the focusing step and the use of single-ion monitoring scan mode are responsible for improving detection limits.     

Separation of polypeptides by isoelectric point focusing in electrospray-friendly solution using a multiple-junction capillary fractionator

We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.     

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.     

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.     

Development of non-gel-based two-dimensional separation of intact proteins by an on-line hyphenation of capillary isoelectric focusing and hollow fiber flow field-flow fractionation

A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (FlFFF). The method developed here (CIEF-HFFlFFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFFlFFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.     

Simultaneous Determination of Ionization Constants and Isoelectric Points of 12 Hydroxy-s-Triazines by Capillary Zone Electrophoresis and Capillary Isoelectric Focusing

Capillary zone electrophoresis (CZE) was used to separate and determine simultaneously the pK(1), pK(2), and pI values of 12 environmentally relevant hydroxytriazines (hydroxymetabolites of atrazine, terbuthylazine, simazine, and propazine and four (arylamino)-s-triazines) and observe the effects of the alkylamino and arylamino substituents on the measured values. Capillary isoelectric focusing (CIEF) methods were developed to measure the pI of these compounds and compare those values with the CZE-measured pI's. CZE and CIEF can provide accurate pK and pI values reasonably fast, and pI values measured by the two techniques agree. Knowledge of the pK and pI values of hydroxytriazines is important for an understanding of the binding mechanisms of these molecules in environmental matrices. Because the hydroxytriazines may exist as a myriad of species [Formula: see text] neutral, charged, zwitterionic, and keto-enol tautomeric [Formula: see text] depending on structure and pH, we briefly addressed the existence of these species relative to their electrophoretic analysis.     

Sensitive and universal indirect chemiluminescence detection for capillary electrophoresis of cations using cobalt(II) as a probe ion

Highly sensitive and universal indirect chemiluminescence detection for capillary electrophoresis of cations was described. This novel method is based on use of the ultrasensitive cobalt(II) as a probe ion in the running buffer. A strong and stable background chemiluminescent signal can be generated by the luminol-hydrogen peroxide reaction catalyzed by cobalt(II) ion. Displacement of the cobalt(II) probe ion in the running buffer by a migrating sample cation results in a quantifiable decrease in the background signal. The conditions for electrophoresis and the chemiluminescent reaction were systematically investigated using a commercial capillary electrophoresis instrument with an in-house-built chemiluminescence detector. Under the optimal conditions, the detection limits of the concentration for manganese(II), cadmium(II), nickel(II), lead(II), and 14 lanthanides were (3.0-6.0) x 10(-9) mol/L (S/N = 3), which was approximately 3 orders of magnitude better than indirect UV detection and 2 orders better than indirect laser-induced fluorescent detection. A mixture of 18 metal ions including 14 lanthanides was efficiently separated within 3.5 min using lactate to partially complex the metal ions. Our data demonstrated that CE with indirect CL detection was a powerful and universal tool for analysis of inorganic and organic cations.     

Dynamic kinetic capillary isoelectric focusing: a powerful tool for studying protein-DNA interactions

A new method called dynamic kinetic capillary isoelectric focusing (DK-CIEF) is presented for the study of protein-DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein-DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants.     

Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection

A capillary isoelectric focusing (CIEF) system with liquid core waveguide (LCW) laser-induced fluorescence whole column imaging detection was developed in this study. A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated LCW. The excitation light was introduced at one end of the capillary, and propagated forward within the capillary. As the Teflon AF 2400 capillary has a refractive index (n = 1.29-1.31) lower than that of water (n = 1.33), total internal reflection was very apparent The employment of the Teflon AF 2400 capillary avoided the use of high refractive index additives such as glycerol, accommodating the system to wider applications. Due to its inert chemical properties, the capillary exhibited limited protein adsorption and electroosmotic flow; thus, the need for capillary preconditioning with polymeric solution and the addition of polymeric additives into the sample mixture can be avoided. Three types of proteins, naturally fluorescent proteins, covalently labeled proteins, and noncovalently labeled proteins, were examined using this method. CIEF under denaturing conditions was also explored, and several advantages over the native mode were found. When compared to a commercially available instrument with UV detection, the separation efficiency and peak capacity were similar while the detection sensitivity was enhanced by 3-5 orders of magnitude.     

Evaluating preparative isoelectric focusing of complex peptide mixtures for tandem mass spectrometry-based proteomics: a case study in profiling chromatin-enriched subcellular fractions in Saccharomyces cerevisiae

We have evaluated the use of free-flow electrophoresis, an emerging separation method for preparative isoelectric focusing of complex peptide mixtures, as a tool for high-throughput tandem mass spectrometry-based proteomic analysis. In this study, we investigated the ability of free-flow electrophoresis to resolve and fractionate complex peptide mixtures and also the effectiveness of using peptide isoelectric point in conjunction with peptide match probability scoring in sequence database searching. As a model system for this study, we analyzed a chromatin-enriched fraction from the yeast Saccharomyces cerevisiae. This mixture was fractionated using preparative isoelectric focusing by free-flow electrophoresis, followed by online capillary liquid chromatography electrospray tandem mass spectrometry and sequence database searching. Our results demonstrate that (1) FFE effectively resolves and fractionates complex peptide mixtures on the basis of peptide isoelectric point and (2) the introduction of peptide pI is effective in minimizing both false positive and false negative sequence matches in sequence database searching of tandem mass spectrometry data.