Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Matrix-assisted laser desorption/ionization mass spectrometry of synthetic polymers. 7. analysis of fatty alcohol ethoxylates by coupled liquid chromatography at the critical point of adsorption and MALDI-TOF mass spectrometry

Fatty alcohol ethoxylates can be analyzed efficiently with respect to functionality and molar mass by coupled liquid chromatography and MALDI-TOF mass spectrometry. Both techniques are coupled via a robotic interface, where the matrix is coaxially added to the eluate and spotted dropwise onto the MALDI target. It is shown that liquid chromatography at critical conditions of adsorption coupled to MALDI-TOF yield useful structural information on oligomer masses and chemical composition. In particular, the analysis of technical fatty alcohol ethoxylates by LC-CC/MALDI-TOF reveals the presence of different functionality fractions in one sample. The oligomer distributions of all functionality fractions are determined.     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

Protein profiling by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

An automated system for intact protein analysis is described that combines capillary isoelectric focusing (CIEF), reversed-phase liquid chromatography (RPLC), and electrospray ionization-mass spectrometry (ESI-MS). Performance is demonstrated with a complex yeast enzyme concentrate. CIEF is performed with a microdialysis membrane-based cathodic cell that permits pI fractions to be sampled and stored for subsequent LC-MS analysis. A total of 50 microg protein is loaded onto the capillary. Ten fractions are stored which span the pI range 3-10. Each fraction is subsequently cleaned on a reversed-phase trap column and then characterized by LC-MS. MaxEnt1 is used to deconvolute the raw mass spectra to obtain the molecular weight (MW) of intact proteins/peptides in the sample. A two-dimensional display of pI vs. MW is illustrated for the 500 most prevalent species as identified by MaxEnt1.     

Poly(glycidyl methacrylate-divinylbenzene) based immobilized pH gradient capillary isoelectric focusing coupling with MALDI mass spectrometry for enhanced neuropeptide analysis

Herein, we report an immobilized pH gradient (IPG) capillary isoelectric focusing-matrix-assisted laser desorption/ionization mass spectrometry (CIEF-MALDI MS) platform designed for the separation of complex neuropeptides. This platform features a poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB)-based monolithic column for CIEF separation. Different from regular CIEF, carrier ampholytes are preimmobilized on the monolithic surface instead of being added to the sample. An off-line coupling of IPG-CIEF to MALDI MS has been established. Comparison with regular CIEF and optimizations are performed with bovine serum albumin tryptic peptides and extracted neuropeptide mixtures from crustacean Callinectes sapidus. It has been demonstrated that the separation of complex peptide mixtures in neutral and basic pH ranges can be achieved in less than 10 min with comparable separation efficiency with regular CIEF, while the MS signal is significantly enhanced when employing IPG-CIEF. Enhanced neuropeptide detection is also observed after coupling IPG-CIEF with MALDI MS.     

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2 × 10⁶ mL⁻¹. Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.     

Capillary isoelectric focusing in pseudo-closed channel coupled to matrix assisted laser desorption/ionization mass spectrometry for protein analysis

Capillary isoelectric focusing (CIEF) was performed in pseudo-closed channel to separate proteins on a plastic chip. Pseudo-closed channel provided a novel way to couple protein separation by CIEF to MALDI mass spectrometry without eluting the focused proteins.     

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…     

Use of electrophoretic techniques and MALDI–TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and “washed pellets”

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.     

Performance of capillary gel electrophoretic analysis of oligonucleotides coupled on-line with electrospray mass spectrometry

Synthetic oligonucleotides (ODNs) are routinely analyzed using capillary gel electrophoresis (CGE) for size-sieving based separations as well as electrospray mass spectrometry (ESI-MS) for identification. On-line coupling of these methods is therefore desired in order to combine the analytical capabilities provided by both methods. Performance of on-line CGE-ESI-MS systems is influenced by various parameters, and choice of optimal conditions is crucial for successful coupling experiments. In this study, we explore characteristics of the on-line coupled CGE-ESI-MS system for ODN analysis. Effects of CGE buffer concentration, capillary length, separation and orifice voltage on CGE separation and MS detection of a phosphodiester ODN mixture were examined. Attention was paid to the influence of the interface, such as geometry of capillary alignment, sheath liquid flow-rate and sheath liquid composition on performance of the system.     

Hyphenation of capillary high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for nano-scale screening of single-bead combinatorial libraries

This paper focuses on the technical aspects of chemical screening from 384-well plate nano-scale single-bead combinatorial libraries. The analytical technique utilized is a combination of capillary liquid chromatography with ultraviolet detection and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The HPLC/MALDI-MS hyphenation is achieved by means of a micro-fraction collector with a peak detection system that automatically collects the peaks onto the MALDI targets for subsequent characterization. Several experimental parameters such as type of 384-well plate, well-plate sealing foils, and a column-switching procedure were investigated using a small test library of nine components. Additionally, the influence of different MALDI matrices, different MALDI targets and sample-spotting techniques on the MALDI detection sensitivity as well as the ruggedness and sample throughput capacity of this technique were studied. Optimum results for the analytes investigated were obtained with 2,5-dihydroxybenzoic acid using on-line mixing of HPLC effluent and matrix solution. To demonstrate the potential of this capillary HPLC/MALDI-TOFMS method, its application to several single-bead libraries was investigated. The instrumental method allowed for the rapid identification and purity assessment of combinatorial libraries with detection limits down to the higher femtomole level using both UV detection and MALDI mass spectrometry.     

Complex analytical approach to characterization of the influence of carbon dioxide concentration on carbohydrate composition in Norway spruce needles

Water-soluble non-structural carbohydrates (NSC) in the needles of Norway spruce Picea abies [L.] Karst have been studied by using a combination of several separation techniques, having various detectors, with mass spectrometry. The intent was to find a suitable methodology that enables the characterization and determination of NSC, covering a wide range of molar masses, and being suitable to assess how NCS are influenced by both external conditions, e.g. different carbon dioxide (CO(2)) concentrations, light intensity, and by internal conditions such as the needle age. The techniques were liquid-liquid extraction, high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), asymmetrical flow field-flow fractionation (AsFlFFF), electrospray ionization mass spectrometry (ESI-MS), and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). NSC were extracted by a methanol/chloroform/water mixture into the water-rich phase. Application of AsFlFFF and SEC, using refractive index (RI) and multi-angle light scattering (MALS) detectors to the water-rich extracts resulted in three or four main fractions covering molar masses from 10(3) to 10(6)g/mol. Individual fractions collected from SEC were directly subjected to both MALDI and ESI-MS analysis in order to identify NSC. MALDI mass spectra confirmed the presence of hexose oligomers in individual fractions while ESI-MS was used for evaluation of low mass NSC. HPLC-RI was used for quantification of NSC and predominant carbohydrates were found to be fructose, glucose, and sucrose. The changes in their content during seasonal course were studied in detail. HPLC coupled to ESI-MS enabled the identification of low concentration NSC like raffinose that occurred in the needles of autumn samplings. An influence of the increased CO(2) concentration on sucrose and glucose accumulation was observed and it was found that the light intensity as well as the needle age has significant influence on the sucrose content.     

Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics

This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30 mm × 0.32 mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.     

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.     

A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B).     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection

A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.     

Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.     

Capillary electrophoresis-mass spectrometry for impurity profiling of basic pharmaceuticals using non-volatile background electrolytes

A generic approach has been developed for coupling capillary electrophoresis (CE) using non-volatile background electrolytes (BGEs) with mass spectrometry (MS) using a sheath liquid interface. CE-MS has been applied for basic and bi-functional compounds using a BGE consisting of 100 mM of TRIS adjusted to pH 2.5 using phosphoric acid. A liquid sheath effect is observed which may influence the CZE separation and hence may complicate the correlation between CE-UV and CE-MS methods. The influence of the liquid sheath effect on the migration behavior of basic pharmaceuticals has been studied by simulation experiments, in which the BGE outlet vial is replaced by sheath liquid in a CE-UV experiment. As a consequence of the liquid sheath effect, phosphate based BGEs can be used without significant loss of MS sensitivity compared to volatile BGEs. The use of buffer constituents such as TRIS can lead to lower detection limits as loss of MS sensitivity can be compensated by better CE performance. TRIS based BGEs permit relatively high injection amounts of about 100 pmol while maintaining high resolution. The ESI-MS parameters were optimized for a generic method with maximum sensitivity and stable operation, in which the composition of the sheath liquid and the position of the capillary were found to be important. Furthermore, the nebulizing pressure strongly influenced the separation efficiency. The system showed stable performance for several days and a reproducibility of about 15% RSD in peak area has been obtained. Nearly all test compounds used in this study could be analyzed with an MS detection limit of 0.05% measured in scan mode using extracted ion chromatograms. As a result, CE-MS was found to be a valuable analytical tool for pharmaceutical impurity profiling.     

毛细管电泳-电喷雾质谱联用技术及其在蛋白质分析领域中的应用

综述了毛细管电泳与电喷雾质谱联用的接口技术、分离模式及其在蛋白质分析领域中的应用，特别是毛细管等电聚焦与电喷雾质谱联用在蛋白质组学中研究进展。