Combination Of Capillary Electrophoresis Research Articles

Application of sequential paired covariance to liquid chromatography-mass spectrometry data enhancements in both the signal-to-noise ratio and the resolution of analyte peaks in the chromatogram

The algorithm of sequential paired covariance (SPC) has been previously reported to dramatically enhance the signal-to-noise ( S/N) ratio for on-line separations combined with mass spectrometry. That initial study focused on a limited number of data sets derived from the combination of capillary electrophoresis (CE) with time-of-flight mass spectrometry using an electrospray interface. Results from the initial study clearly demonstrated that a significant enhancement (almost two orders of magnitude) in the S/N ratio of the eluting peaks in the electropherogram could be obtained, facilitating identification of the analytes. In this report, the algorithm has been applied to liquid chromatography-mass spectrometry data obtained on a triple quadrupole instrument and we have evaluated the general applicability of the SPC approach to several types of microcolumn separations with mass spectrometric detection, including CE coupled with Fourier transform ion cyclotron resonance mass spectrometry. In all the cases we tested, we found the algorithm enhanced the S/N ratios of the resulting chromatograms or electropherograms to a similar extent. This report further demonstrates the SPC approach to enhance the resolution as well as the S/N ratio of the eluting peaks of a complex peptide mixture. While many variations of the algorithm are possible, we have also found higher order covariance (e.g., 3rd order) is useful for eliminating coincidental noise in sequential mass spectra, giving the potential to extract broad, low intensity analyte peaks. We also demonstrate the sequential covariance approach for enhancing the S/N ratio of mass spectra.

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.

High performance fourier transform ion cyclotron resonance mass spectrometric detection for capillary electrophoresis

AbstractWe describe the current state of the on‐line combination of capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS), and discuss aspects of the FTICR technique relevant to its use as a detection scheme for on‐line separations. Aspects including sensitivity, mass resolution, duty cycle, and tandem mass spectrometric capabilities are discussed in the context of online separations with examples from the authors' laboratory.

Analysis of a standard peptide mixture by capillary electrophoresis with mass spectrometry and with tandem mass spectrometry.

The combination of capillary electrophoresis with mass spectrometry allows efficient separation and identification of components in mixtures with greater specificity than can be obtained using capillary electrophoresis and ultra violet detection alone. For the mixture of peptides analysed in this application, molecular mass information was obtained on all of the components in the initial analysis. On-line capillary electrophoresis/tandem mass spectrometry was then used to generate amino acid sequence information for each of these peptides, at the pmole level, in the form of collision-induced production-ion spectra which were recorded only over the relevant time windows.

Separation of twenty‐one naphthalene sulfonates by means of capillary electrophoresis

AbstractThe production and use of naphthalene sulfonates can easily cause pollution of surface and other types of waters. In the present study, capillary electrophoresis in combination with UV absorption detection was used to separate 21 amino‐ and hydroxy‐substituted naphthalene sulfonates which included multiple isomeric compounds. The influence of various parameters such as pH (which turned out to be extremely important), temperature of the surrounding air flow, and the use of buffer additives (micelles, cyclodextrins, organic modifiers) was studied. Complete separation of all analytes including the isomers, was achieved in two runs with a 50 mM boric acid/borate buffer, containing either 100 mM sodium dodecylsulfonate or 15% acetonitrile. The limits of detection obtained for the individual compounds typically were 20μgI−1. River water samples spiked at this concentration level could be analysed using a simple three‐step sample clean‐up procedure.

Application of Sequential Paired Covariance to Capillary Electrophoresis Electrospray Ionization Time-of-Flight Mass Spectrometry: Unraveling the Signal from the Noise in the Electropherogram

The combination of capillary electrophoresis (CE) with electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) has recently been demonstrated. When CE is combined with TOF-MS using an electrospray interface, the method provides fast, high-resolution separations with rapid mass analysis and the potential for very high sensitivity. In analyzing the data, one first reconstructs an electropherogram from the data set and then identifies peaks in the mass spectra. The mass spectra corresponding to the peaks in the electropherogram are then inspected to enable solute identification. However, background noise often prevents detection of peaks in the reconstructed electropherogram, thus interfering with the analysis. In this work we demonstrate alternative electropherogram reconstruction techniques that enhance its signal-to-noise ratio, allowing more immediate identification of the number of components in a mixture and their corresponding retention times. The techniques described here should also be adaptable for on-line analysis of CE-ESI-TOF data and the combination with other separation techniques coupled to mass spectrometry (e.g., LC/MS), as well as any multichannel detection scheme. 21 refs., 4 figs., 1 tab.

Capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for direct analysis of cellular proteins.

The combination of capillary electrophoresis (CE) with electrospray ionization (ESI) mass spectrometry has proven to be broadly applicable to a wide range of biologically important compounds. When combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, the combined method, in addition to high-resolution separations, affords high-resolution precision mass measurements for analytes separated from complex mixtures. Direct chemical analysis of single cells has received considerable attention in recent years; the single cell approach provides a major step toward answering important questions in the field of cellular biochemistry. In this work we present preliminary results which demonstrate the feasibility of using the CE-ESI-FTICR combination as a high-performance detection scheme for the analysis of cellular proteins acquired directly from small populations (i.e., 5-10) of intact living cells. The human erythrocyte was chosen as a model system owing to its availability, relatively homogeneous composition, and thorough documentation of contents by previous researchers. In this work we demonstrate the on-line acquisition of high-resolution mass spectra (average resolution > or = 45,000 fwhm) of both the alpha and the beta chains of hemoglobin acquired from the injection of 10 human erythrocytes (corresponding to 4.5 fmol of hemoglobin). Given the extremely small volume of the human erythrocyte (typically 87 fl/cell), the techniques implemented here should also be adaptable to the study of larger mammalian cell systems.

Capillary electrophoresis—matrix-assisted laser-desorption ionization mass spectrometry of proteins

An “off-line” combination of capillary electrophoresis (CE) with matrix-assisted laser-desorption mass spectrometry (MALDI-MS) has been developed for the structural characterization of CE-separated peptides and proteins. Using a sheath flow interface, similar to that developed for “on-line” CE—fast atom bombardment MS and CE—electrospray MS, an efficient sample isolation procedure has been developed which is applicable to bioorganic compounds in aqueous buffer solutions. This isolation procedure, with subsequent transfer to the MALDI-MS sample target, has been successfully used for the direct analysis of CE-separated proteins of M r up to 67 000, and a mixture of apolipoprotein AII monomer and homodimer, using sample amounts of less than 1 pmol.

Structural chacracterization of polypeptides and proteins by combination of capillary electrophoresis and 252Cf plasma desorption mass spectrometry

An efficient and sensitive method for the isolation and transfer of peptides and proteins from capillary zone electrophoresis separation for subsequent analysis by 252Cf plasma desorption mass spectrometry was developed. Sample isolation on to nitrocellulose-coated targets for mass spectrometric analysis is performed by using a stainless-steel microtube pre-filled with aqueous buffer solution, to which the capillary end is connected, and the peptide is collected by applying a suitable transfer voltage according to the separation voltage. Low- and sub-picomolar sample amounts were isolated with high transfer efficiency and reproducibility, without the necessity for independent determination of electroosmotic flow-rates. Plasma desorption mass spectra of several peptides and proteins showed predominantly intact molecular ions; however, for several peptides partial oxidative modification was found which can be accounted for by the electrophoretic separation and/or transfer conditions. First applications to peptides and proteins show the feasibility of this off-line combination for primary structure characterization, such as by in situ chemical modification and enzymatic proteolysis reactions on the sample target prior to mass spectrometric analysis.

New developments in biochemical mass spectrometry: electrospray ionization

The principles, development, and recent application of electrospray ionization-mass spectrometry (ESI-MS) to biological compounds are reviewed. ESI-MS methods now allow determination of accurate molecular weights for proteins extending to over 50,000, and in some cases well over 100,000. Similar capabilities are being developed for oligonucleotides. The instrumentation used for ESI-MS is briefly described and it is shown that, although ionization efficiency appears to be uniformly high, detector sensitivity may be directly correlated with molecular weight. The use of tandem mass spectrometry (e.g., MS/MS) for extending collision-induced dissociation (CID) methods to the structural studies of large molecules is described. For example, effective CID of various albumin species (molecular weight approximately 66,000) can be obtained, far larger than obtainable for singly charged molecular ions. The combination of capillary electrophoresis, in both free solution zone electrophoresis and isotachophoresis formats, as well as microcolumn liquid chromatography with ESI-MS, provides the capability for on-line separation and analysis of subpicomole quantities of proteins. These and other new developments related to ESI-MS are illustrated by a range of examples. Fundamental considerations suggest even more impressive developments may be anticipated related to detection sensitivity and methods for obtaining structural information.

The achievement and uses of ultra-high vacuum: L. Holland, New Scientist, 10 (232), 27 April 1961, 188–191

The adsorption of chemically similar but differently oxygen reactive phenolic-acid derivatives on the Ti-nanotubes (TiNTs) surfaces to increase and/or broaden their photo-induced activity was studied using Raman and X-ray photoelectron spectroscopies combined with zeta-potential analyses. Photo-catalytic activities and stabilities of newly synthesized particles were evaluated by using high-resolution capillary electrophoresis in combination with cyclic voltammetry and spin-trapping EPR spectroscopy.The modification with caffeic acid (CA) resulted in well-oriented and dense but oxygen semi-stable thin layer (1–3 nm) of self-assembled mono-molecular and/or bi-dentate coordinated molecules on the TiNTs’ surfaces, which narrowed the band gap from 2.9 eV (for un-modified TiNTs) to 1.55 eV, but however restrict the hydroxyl radicals generation under both UV (320 nm) and VIS (450 nm) source radiations. On the other hand, the gallic acid (GA) resulted in situ polymerized GA layer through bi-dentate binding as highly-oxygen-stabilized surface structure, yielding narrower band gap of 2.25 eV and increased hydroxyl radical’s generation under both exposure lights. The third tested hydroxybenzoic acid (HA), resulted to an unstable layer bonded thorough single-hydrogen bonding mechanism.This work offers a new modification strategy for stable (oxygen and photo-induction related) and highly visible–light responded TiNTs as photocatalyst.