Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

Recent developments in capillary electrophoresis-mass spectrometry of proteins and peptides.

Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.     

On-target fraction collection for the off-line coupling of capillary isoelectric focusing with matrix-assisted laser desorption/ionization mass spectrometry.

In the present work, we describe a collection system for the off-line coupling of capillary isoelectric focusing (CIEF) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this system, the capillary effluent is directly deposited in fractions onto the MALDI target via the use of a sheath liquid. The collected fractions are subsequently supplemented with matrix and further analysed by MALDI-TOF mass spectrometry for mass assignment. The experimental set-up includes a fiber optic based UV detector operating at 280 nm, which allows the study of the influence of the sheath liquid composition on the CIEF separation. The influence of the carrier ampholyte concentration on the protein MALDI spectra was also evaluated and the feasibility of the collection method was finally demonstrated with a mixture of four standard proteins.     

Multi-purpose capillary electrophoresis system with concentration gradient detection

A robust, inexpensive and versatile capillary electrophoresis (CE) system for routine and rapid analysis is reported, which consists of a rugged cartridge holding a 20-mum i.d. 15-cm long capillary, and an inexpensive, universal and sensitive concentration gradient detector. The design of the cartridge simplifies the sample introduction process and makes it possible to perform many separation modes, including moving boundary capillary electrophoresis (MBCE), capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF), on the same system. This arrangement provides more information about a sample's components since analytes can be separated by different modes performed on the same CE system. The detector only consists of a low-power HeNe laser, or laser diode, and a photodiode position sensor. Amino acids and proteins of 10(-6)-10(-3)M concentration can be separated by different capillary electrophoretic modes, and detected directly by the detector. The universal detector shows particularly good sensitivity when applied to CE separation modes having self-concentration and focusing effects. Femtomoles of proteins were separated and detected with CIEF. In addition, a short and narrow capillary allows use of high electrical fields which facilitate rapid separations. Four amino acids at millimolar concentrations were fully separated and detected in less than 80 sec by the MBCE mode when a high electric field was applied. The physical size of the whole system is much smaller than that of conventional CE instruments with UV absorbance or fluorescence detector.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

用于蛋白质分析的毛细管等电聚焦-电喷雾质谱接口的改进

蛋白质组学对其分析技术提出了大规模、高通量的要求 [1] .传统的等电聚焦 ( p I) -分子量 ( MW)双向电泳技术 ( 2 D- Gel)尽管在蛋白质组学研究中占有重要地位 ,但其操作繁杂、工作周期长 .Pandey等[1] 将毛细管等电聚焦 ( CIEF)与电喷雾质谱 ( ESIMS)联用 ,使得 p I和 MW两维分离鉴定技术变得简单迅速 .但 CIEF- MS的接口操作需中断高压和将毛细管阴极端插入电喷雾管 ,故引起分析蛋白质的散焦和不重现 .本工作改进了 CIEF- MS接口 ,采用毛细管阴极端和电喷雾针一体化的电喷雾接口 ,无需中断高压 ,实现了 CIEF- MS的在线联…     

Miniaturized capillary isoelectric focusing in plastic microfluidic devices

We report the demonstration of miniaturized capillary isoelectric focusing (CIEF) in plastic microfluidic devices. Conventional CIEF technique was adapted to the microfluidic devices to separate proteins and to detect protein-protein interactions. Both acidic and basic proteins with isoelectric points (pI) ranging from 5.4 to 11.0 were rapidly focused, mobilized, and detected in a 1.2 cm long channel (50 microm deep x 120 microm wide) with a total analysis time of 150 s. In a device with a focusing distance of 4.7 cm, the separation efficiency for a basic protein, lysozyme, was achieved as high as 1.5 x 10(5) plates, corresponding to 3.2 million plates per meter. We also experimentally confirmed that IEF resolution is essentially independent of focusing length when the applied voltage is kept the same and within a range that it does not cause Joule heating. Further, we demonstrated the use of miniaturized CIEF to study the interactions between two pairs of proteins, immunoglobulin G (IgG) with protein G and anti-six histidine (anti-6xHis) with 6xHis-tagged green fluorescent protein (GFP). Using this approach, protein-protein interactions can be detected for as little as 50 fmol of protein. We believe miniaturized CIEF is useful for studying protein-protein interactions when there is a difference in pI between a protein-protein complex and its constitutent proteins.     

Miniaturization of capillary isoelectric focusing.

Miniaturization of whole-column imaging capillary isoelectric focusing (CIEF) is discussed. A 1.2 cm capillary was used as a separation column for CIEF. The experimental results for the analysis of two pI markers and the protein myoglobin showed that good CIEF separation results could be obtained. Secondly, a light-emitting diode (LED) was used as the light source for the whole-column absorbance imaging detection. The focusing of both the pI markers and myoglobin were observed with the LED light source. The whole-column imaging CIEF instrument was simplified and miniaturized by the use of the LED. Further developments are also discussed.     

Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation

On-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed using a microinjector as the interface for performing two-dimensional (2-D) protein/peptide separations of complex protein mixtures. The focusing effect of CIEF not only contributes to a high-resolution protein/peptide separation, but also may permit the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. The preparative capabilities of CIEF are much larger than most of capillary-based electrokinetic separation techniques since the entire capillary is initially filled with a solution containing proteins/peptides and carrier ampholytes for the creation of a pH gradient inside the capillary. The focused peptides which have a similar pI are coinjected into the second separation dimension and further resolved by their differences in hydrophobicity. The resolving power of combined CIEF-CRPLC system is demonstrated using the soluble fraction of Drosophila salivary glands taken from a period beginning before steroid-triggered programmed cell death and extending to its completion. The separation mechanisms of CIEF and CRPLC are completely orthogonal and the overall peak capacity is estimated to be around approximately 1800 over a run time of less than 8 h. Significant enhancement in the separation peak capacity can be realized by further increasing the number of CIEF fractions and/or slowing the solvent gradient in CRPLC, however, at the expense of overall analysis time. The results of our preliminary studies display significant differences in the separation profiles of peptide samples obtained from salivary glands of animals staged at the 6 and 12 h following puparium formation.     

Identification of kappa and lambda chains of the major immunoglobulin G subclasses by capillary zone electrophoresis

Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains. Various analytical conditions and CE modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC) have been thoroughly studied. CZE was found to be the most convenient way to separate IgG subclasses. Three of the human IgG subclasses were resolved using uncoated fused-silica and 50 mM phosphate, pH = 9.3, as operating buffer at 20 kV and detection at 214 nm. IgG1kappa was completely separated from IgG2kappa and IgG3kappa, whereas IgG2kappa co-migrated with IgG4kappa, which is the minor IgG subclass. Under the same conditions IgG4lambda was completely separated from IgG1lambda, IgG2lambda and IgG3lambda, enabling the identification of the various lambda chains. The developed CE method is rapid and can be applied to the identification of the major immunoglobulin G subclasses in respect to their variable kappa and lambda chains.     

Application of nanoscale packed capillary liquid chromatography (75 μm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics

Nanoscale separation techniques, nanoscale packed capillary columns (75 μm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nanoscale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.     

Comprehensive two-dimensional chromatography and capillary electrophoresis coupled with tandem time-of-flight mass spectrometry for high-speed proteome analysis

A comprehensive two-dimensional capillary liquid chromatography and capillary zone electrophoresis system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) proteomics analyzer is presented. Protein/peptide samples were separated by capillary high-performance liquid chromatography (cHPLC). The effluents from cHPLC (the first dimension) were continuously transferred into capillary zone electrophoresis (CZE, the second dimension) through a novel valve-free hydrodynamic sampling interface. The CZE effluents were mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix sheath flow via CE-MALDI interface, and then directly deposited on the MALDI target at a 3 s time-interval for further MS analysis. The high efficiency of the overall system was demonstrated by analysis of proteins in D20 (human hepatocellular carcinoma model in nude mice with high metastatic potential) liver cancer tissue. More than 300 proteins were identified, which proved the system potential for high-throughput analysis and application in proteomics.     

Profiling and screening analysis of 27 aromatic amino acids by capillary electrophoresis in dual modes

An efficient capillary electrophoretic (CE) profiling and screening system based on dual modes of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) was developed for the simultaneous determination of 23 nonprotein amino acids (NPAAs) and 4 protein amino acids with aromatic moiety. It involves separation by an uncoated fused-silica capillary under phosphoric acid buffer in CZE mode and by another uncoated fused-silica capillary under neutral sodium dihydrogen phosphate buffer containing sodium dodecyl sulfate in MEKC mode. Migration orders of the amino acids studied on the two separation modes under each optimum condition were very different. The repeatability of migration times measured by the CZE and MEKC was found to be better than 4.8 and 3.4%, respectively, thereby enabling to cross-check the identification of each amino acid. The method linearity and limit of detection of the CZE for each amino acid were found to be adequate for the assay of aromatic amino acids. When the present CE profiling and screening analysis in dual modes was applied to plant seeds, NPAAs such as mimosine from Mimosa pudica Linné, and 2-phenylglycine from Lindera erythrocarpa Makino were positively detected along with tryptophan, phenylalanine and tyrosine.     

Comprehensive two dimensional separation based on coupling micellar electrokinetic chromatography with capillary isoelectric focusing

Concentrating properties of the Capillary Isoelectric Focusing (CIEF) system with continuous whole-column-imaging detection were investigated for application as a second dimension in a comprehensive two-dimensional (2D) separation process. The concentration/separation/detection was completed within 4 min in a 300 microm inner diameter capillary. As the key to the successful coupling of CIEF to a first dimension separation, a novel interface was developed. A 10-port valve with two conditioning loops was used to perform both comprehensive collection and dialysis desalting of the first dimensional effluent, and as an interface coupling Micellar Electrokinetic Chromatography (MEKC) to CIEF. In the loop, salt and other unwanted first dimension effluent components were eliminated by dialysis and carrier ampholytes (CAs) were added. Peak broadening during the dialysis did not have significant impact on the CIEF separation because of its concentrating effect. Protein digests were first separated by MEKC followed by isoelectric point (pI) using whole-column-imaged CIEF. The dialysis interface allows general coupling of the whole-column-imaged CIEF to microscale separations.     

Capillary isoelectric focusing--reproducibility and protein adsorption

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3-15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long-time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self-amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)-coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused-silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.     

Application of capillary isotachophoresis-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

By employing a capillary ITP (CITP)/CZE-based proteomic technology, a total of 1795 distinct mouse Swiss-Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss-Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF-based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2-database reference set.     

A beveled tip sheath liquid interface for capillary electrophoresis-electrospray ionization-mass spectrometry

A simple and durable sheath liquid interface for capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) has been developed. This interface utilized a beveled tip emitter and was found to be more sensitive than the conventional sheath liquid interface. The use of a beveled tip reduces the optimal flow rate and therefore decreases sample dilution. The interface utilized a 380 microm inner diameter and 400 microm outer diameter beveled tapered tip. Because of the large inner diameter and outer diameter of the tip, the interface is robust and can be easily implemented. The performance of this interface for CZE-ESI-MS and micelle electrokinetic capillary electrophoresis-electrospray-mass spectrometry, as demonstrated by the analysis of synthetic drugs and triazine mixtures, was significantly better than results obtained using a conventional sheath liquid interface.     

毛细管等电聚焦-毛细管区带电泳二维蛋白质分离平台的构建及其性能

通常采用二维聚丙烯酰胺凝胶电泳 ( 2 D- PAGE)分析组织或细胞的全蛋白质 [1] ,但难与质谱 ( MS)直接联用 .用高效液相色谱 ( HPLC)和毛细管电泳 ( CE)分离分析蛋白质和多肽的一维分离模式的分辨率和峰容量有限 .多维柱联用技术比一维分离有更高的分辨率和峰容量 [2 ] ,便于和 MS直接联用 [3,4 ] ,易于实现自动化 .目前 ,有关 2 D- CE的报道相对较少 [5～ 7] ,我们初步实现了将 2 D- PAGE由平板转移到毛细管中[6 ,7] ,但凝胶柱的制作烦琐 ,存在交叉污染 ,不能与 MS直接联用 .本文用微透析中空纤维膜为接口构建了毛细管等电聚焦 ( …     

Separations of enantiomers by preparative capillary isotachophoresis.

The use of capillary isotachophoresis (ITP), operating in a discontinuous fractionation mode, for preparative separations of enantiomers of chiral compounds was studied. The ITP separations were carried out in the column-coupling configuration of the separation unit provided with the preseparation column of a 1.0 mm ID and the trapping column of a 0.8 mm ID. Such a configuration of the CE separation unit offers several working regimes suitable to preparative separations of enantiomers. 2,4-Dinitrophenyl-DL-norleucine (DNP-Norleu) was employed as a model analyte in our experiments with beta-cyclodextrin serving in the electrolyte solutions as a chiral selector. The preparative separations lasting about 20 min were evaluated by ITP and (more often) by capillary zone electrophoresis (CZE). It was found that one preparative run provided up to 14 microg of pure DNP-Norleu enantiomers. This corresponded to a 75 times higher production rate of ITP relative to a maximum value of this parameter as estimated for preparative CZE runs in cylindrical capillaries (0.5 pmol/s). About 75% of the DNP-Norleu enantiomers loaded into the preparative equipment could be recovered in pure enantiomer fractions. Contiguous natures of the zones in the ITP stack and adsorption losses of the enantiomers in the isolation step were found to set practical limits for a further enhancement of the recovery rates in the isolation of pure enantiomers.