Åsa Emmer

Improved capillary zone electrophoretic separation of basic proteins, using a fluorosurfactant buffer additive

Abstract In this paper, a new method to reduce the adsorption of basic proteins in capillary zone electrophoresis is described. Small amounts of a cationic fluorosurfactant are added to the running buffer. This leads to a surface charge reversal. Consequently, proteins at a pH below their p I are repelled from the wall. High efficiencies and symmetrical peaks were obtained for a number of model proteins, even when running buffer solutions with a low ionic strength were employed. Reproducibility was excellent. It is believed that the extreme hydrophobic nature of the fluorocarbon chain of the surfactant is a significant factor for the improved performance.

Parallel reactions in open chip-based nanovials with continuous compensation for solvent evaporation

In an earlier report (Litborn, E., Emmer, Å., Roeraade, J., Anal. Chim. Acta 1999, 401, 11—19), we described a technique for performing chemistry in chip‐based vials. A major problem, solvent evaporation, was partially remedied by using a closed humidity chamber. In this paper we report an improved technique for performing parallel reactions in open, 15 nL volume, chip‐based vials. The evaporation of solvent from the reaction fluid was continuously compensated by addition of solvent via an array of microcapillaries. The suitability of the method was demonstrated by performing eight separate peptide maps of myoglobin in parallel, using the three enzymes trypsin, α‐chymotrypsin and endoproteinase Glu‐C. The total amount of myoglobin utilized to perform the eight digests was less than 100 pmol. The corresponding amount of enzymes was ca. 0.1 pmol per reaction. In order to evaluate the operating limits of the technique, a study of the evaporation of solvents from a series of vials with proportionally smaller volumes operated at different temperatures was performed. The results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pL.

Characterization of proteinases from Antarctic krill (Euphausia superba)

Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes, CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060, and 26,260Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260Da, whereas the CPB enzyme masses likely are 33,730 and 33,900Da. The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degrees C and 1-3 degrees C, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.

Capillary electrophoretic separation of acidic and basic proteins in the presence of cationic and anionic fluorosurfactants

Abstract We report the use of mixture of cationic and anionic fluorosurfactants as addtives for free-flow capillary electrophoresis of proteins. Effective deactivation of the capillary wall is obtained, which allows the use of raw fused-silica capillaries. The magnitude and direction of the electroosmotic flow is strongly affected by the composition of the surfactant mixture, and a suggested model for this behaviour in terms of micellation and formation of admicellar surfactant layers is described. By utilizing mixtures of the oppositely charged surfactants, it is possible to separate both positively and negatively charged proteins in the same run. Due to charge interactions of the fluorosurfactants with the proteins, it is possible to tune the separation selectivity, without having to change the buffer strength or pH. This was demonstrated in particular for the model substances myoglobin and ribonuclease, where the order of elution could be reversed, compared to their elution order in a normal buffer system. Another advantage of the fluorosurfactants additives is their effectiveness in low concentrations (

Capillary electrophoresis, combined with an on-line micro post-column enzyme assay

A system for capillary electrophoresis (CE), combined with a micro post-column reactor for on-line enzyme assays is described. Two detectors are employed. The first detector is an on-column UV detector, utilized to monitor the CE separation, while the second detector is monitoring the reaction product which is formed by adding a flow of substrate in the post-column section. Factors affecting the band broadening in the post-column reactor have been investigated. A short reaction distance as well as a high flow-rate of substrate showed to be optimal. A CE separation of glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconic dehydrogenase (6-PGDH) was performed, where the enzyme activity could be monitored after reaction with nicotinamide—adenine dinucleotide phosphate/glucose-6-phosphate. The minimal detectable amount of G-6-PDH was shown to be in the order of 5 · 10−16 Mol (2 · 10−7M).

Chip-based nanovials for tryptic digest and capillary electrophoresis

A method for performing tryptic digests in chip-based open vials with volumes of 15 nl has been developed. Working in an enclosed environment with increased humidity counteracted the evaporation of water from the vials during the reaction. Peptide maps of native myoglobin were obtained with capillary electrophoresis and compared with the results of digests performed in conventional 100-μl volumes. The peptide maps, after reactions performed in conventional plastic vials and chip-based vials, showed similar patterns. The reaction showed to be more efficient in the chip-based vials compared to digests performed in conventional plastic vials. The increased efficiency was probably due to the large differences in surface-to-volume ratios and surface dependent reactions. The lowest amount of myoglobin utilized to perform one single digest in a chip-based vial was 12 ng. The corresponding amount of trypsin used was 0.2 ng.

Performance of Zwitterionic and Cationic Fluorosurfactants as Buffer Additives for Capillary Electrophoresis of Proteins

Abstract In this work, a study has been made of the performance of a zwitterionic fluorosurfactant and mixtures of a zwitterionic and a cationic fluorosurfactant, when used as buffer additives in capillary electrophoresis. Thus, it showed to be possible to change the direction of the electroosmotic flow by changing the pH of the buffer solution. Possible mechanisms for the behaviour of the electroosmotic flow at different pH and when using different surfactant combinations are suggested. High efficiency separations of some model proteins can be obtained, when a mixture of a cationic and a zwitterionic fluorosurfactant is added to the running buffer solution. By changing concentration proportions between the surfactants, a change in separation selectivity is obtained. This procedure provides an alternative way for selectivity tuning in protein separations by capillary electrophoresis.

Wall deactivation with fluorosurfactants for capillary electrophoretic analysis of biomolecules

This paper describes the use of fluorosurfactants as buffer additives for capillary electrophoretic separation of proteins and peptides. Due to fluorosurfactant bilayer formation at the capillary inner wall, the surface charge can be adjusted and even reversed. If the running buffer pH is kept at a level where the proteins have the same sign of charge as the wall, electrostatic repulsion will be obtained. The protein wall adsorption can therefore be reduced and the separation performance can be noticeably increased. The separation performance can also be further improved by including mixtures of different types of fluorosurfactants in the running buffer. The buffer system can accordingly be adapted for a certain separation problem. Mechanisms for the use of fluorosurfactants for wall deactivation in capillary electrophoretic protein separations is discussed in the present work and some examples of applications are also presented.

Separation of pig liver esterase isoenzymes and subunits by capillary zone electrophoresis in the presence of fluorinated surfactants

An attempt was made to separate the isoenzymes and subunits of pig liver esterase by capillary zone electrophoresis. This enzyme is a complex mixture and is strongly adsorbed on a fused-silica capillary. However, by simply adding a cationic fluorosurfactant to the running buffer, adsorption was significantly reduced. The effects of adding a zwitterionic and a neutral fluorosurfactant were also investigated. Large changes in the elution pattern were observed when using different combinations of these additives. Mixtures of different fluorosurfactants added to the running buffer can therefore be utilized in strategies for optimization of the separation selectivity.

Determination of fluoroquinolones in bovine milk samples using a pipette‐tip SPE step based on multiwalled carbon nanotubes prior to CE separation

A simple CE-UV method was developed for the simultaneous determination of ciprofloxacin, norfloxacin, and ofloxacin in milk samples. The optimum separation was obtained using a 20 mM ammonium dihydrogenphosphate solution with 2 mM cetyltrimethylammonium bromide at pH 3.0 as the BGE. Satisfactory resolution for structurally very similar analytes, like norfloxacin and ciprofloxacin, was achieved without including any organic solvent. Milk samples were prepared using a simple/extraction procedure based on acidic protein precipitation followed by an SPE step using only 5 mg of multiwalled carbon nanotubes as the sorbent material. The LODs for the three compounds were between 7.5 and 11.6 μg/L and the RSDs for the peak areas were between 2.6 and 4.9%. The complete method was applied to spiked real milk samples with satisfactory recoveries for all analytes (84-106%).