Optimization of capillary coating by hydroxyethyl methacrylate for capillary zone electrophoresis of proteins

This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water-methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Sample enrichment techniques in capillary electrophoresis: focus on peptides and proteins

Compared to chromatography-based techniques, the concentration limits of detection (CLOD) associated with capillary electrophoresis are worse, and these have largely precluded their use in many practical applications. To overcome this limitation, researchers from various disciplines have exerted tremendous efforts toward developing strategies for increasing the concentration sensitivities of capillary electrophoresis (CE) systems, via the so-called sample enrichment techniques. This review highlights selected developments and advances in this area as applied to the analyses of proteins and peptides in the last 5 years.     

Recent advances in enantiomer separations by affinity capillary electrophoresis using proteins and peptides

Enantiomer separations by capillary electrophoresis (CE), using proteins as chiral selectors--affinity capillary electrophoresis (ACE) with free solutions and capillary electrochromatography (CEC)--with protein immobilized capillaries, are reviewed. The separation principle, recent advances in this field and some interesting topics are presented. In ACE, various enantiomer separations have been already reported using either plasma proteins or egg white ones. Miscellaneous proteins were also explored in the last few years. On the contrary, only a limited number of enantiomer separations have been successfully achieved in CEC. CEC is not yet mature enough to date, and further investigations, such as efficiency, durability and reproducibility of capillaries, will be necessary for the use of routine analyses. The study of enantioselective drug-protein binding is important in pharmaceutical developments. Some applications including high-performance CE/frontal analysis (HPCE/FA) are introduced in this paper.     

Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides

Excellent correlation was observed for the electrophoretic mobilities measured by capillary zone electrophoresis versus q/MW2/3, where q is the calculated charge and MW is the molecular weight. Mobilities of a set of 33 diverse peptides from enzymatic digests and 10 intact proteins were measured for separations at pH 2.35, 8.0, and 8.15 with constant ionic strength, temperature, and viscosity. The correlation suggests that the frictional drag is proportional to the surface area of a sphere that has a volume proportional to the MW. The correlation of electrophoretic mobility with physicochemical properties will facilitate the elucidation of optimum separation strategies for protein and peptide mixtures.     

Dynamic coating for fast and reproducible determination of basic drugs by capillary electrophoresis with diode-array detection and mass spectrometry

The double coating principle of CEofix buffers was evaluated for the analysis of some basic drugs by capillary electrophoresis-diode-array detection (CE-DAD) and capillary electrophoresis-mass spectrometry (CE-MS). The involatile phosphate present in original low pH CEofix, was replaced with formic acid for hyphenation of CE with MS. The double coating produces a substantial and highly reproducible electroosmotic flow (EOF), even at low pH. The rinsing procedure and electrolyte composition were optimized for both CE-DAD and CE-MS. The system was evaluated with the analysis of a mixture of basic drugs and a spiked urine sample enriched by solid-phase extraction (SPE). The R.S.D. values on the migration time and peak area measured for 28 analyses with CE-DAD were below 0.25 and 2.40%, respectively. For CE-MS, the R.S.D. on the migration time was 0.85% or less and the area precision ranged from 5.65 to 14.33% (for seven injections). The LOD with the developed CE-MS method was below 50 ppb for all five drug standards tested.     

Selectivity change in the separation of proteins and peptides by capillary electrophoresis using high-molecular-mass polyethyleneimine

A study of the capillary electrophoretic separations of proteins and peptides using high-molecular-mass polyethyleneimine (PEI) is presented. Experiments were performed in the PEI-coated capillaries together with the use of this polymer as a buffer additive under different separation conditions. The effects of pH and the concentration of PEI in the buffer on the electroosmotic flow and the migration orders of biopolymers were investigated. The use of the cationic polymer offers an alternative for the modification of the separation selectivity and resolution of biopolymers.     

Peptic and tryptic digestion of peptides and proteins monitored by capillary electrophoresis with contactless conductivity detection

The feasibility of monitoring the peptic and tryptic digestion of peptides and proteins with capillary electrophoresis using contactless conductivity detection was investigated. The peptide minigastrin I and the proteins cytochrome c from bovine heart, human serum albumin (HSA), myoglobin, and bovine serum albumin (BSA) were digested off-line with pepsin, and the resulting peptide and amino acid fragments were successfully separated and detected by conductivity measurement. Cytochrome c and myoglobin were also subjected to off-line cleavage with trypsin. On-line digestion using the electrophoretically mediated microanalysis (EMMA) approach was demonstrated with cytochrome c and apomyoglobin using trypsin.     

Physico-chemical characterization of proteins by capillary electrophoresis

The electrophoretic mobility of proteins was successfully determined by means of capillary electrophoresis (CE) with various background electrolytes (BGEs). The objective was focused on the variation in BGE physico-chemical composition and the consequential impact on the observed protein charge. Experimental and calculated mobilities, according to Henry's equation, versus ionic strength have been compared. For positively-charged lysozyme, a good agreement between observed and calculated mobilities was observed using triethanolamine chloride at pH 7.0 as the BGE. Mobility close to zero was shown using borate (pH 8.0) and phosphate (pH 7.0) at a low ionic strength of about 20 mmol l⁻¹, and as a consequence, specific adsorption of oxyanions was evidenced. Lysozyme retention in the case of reversed-phase high-performance liquid chromatography (RP-HPLC) was decreased by the presence of phosphate ions. CE and HPLC are complementary tools for characterizing the behaviour of lysozyme. On the other hand, the mobility of the negatively-charged α-lactalbumin remained constant as regards phosphate at pH 7.0 in the 20–200 mmol l⁻¹ range, contrary to the decrease that had been expected with the increasing ionic strength. β-Lactoglobulin exhibited increasingly lower mobilities than those expected of boric acid/borate at pH 7.0 and 8.0 (I=20 mmol l⁻¹).     

Direct analysis of peptides and amino acids from capillary electrophoresis

Capillary zone electrophoresis of peptides on a preparative scale and direct analysis of both amino acid sequences and compositions are demonstrated. Using native and synthetic peptides prepared by repetitive runs and appropriate collections, sufficient material for direct sequence analysis is obtained after just a few runs, while single runs suffice for total compositions. Electrophoretic direct amino acid analysis without derivatization is also possible and gives especially high sensitivity for aromatic residues (less than 200 fmols) plus convenient distinction of amidated versus free C-terminal residues after carboxypeptidase digestions.     

Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

Antimicrobial peptides (AMPs) are regarded as attractive alternatives to conventional antibiotics, but their production in microbes remains challenging due to their inherent bactericidal nature. To address these limitations, we have developed a novel AMP fusion protein system based on an encapsulin nanocompartment protein and have demonstrated its utility in enhancing expression of HBCM2, an AMP with activity against Gram-negative bacteria. Here, HBCM2 was fused to the N-terminus of several Encapsulin monomer (Enc) variants engineered with multiple TEV protease recognition site insertions to facilitate proteolytic release of the fused HBCM2. Fusion of HBCM2 to the Enc variants, but not other common carrier proteins, enabled robust overexpression in Escherichia coli C43(DE3) cells. Interestingly, variants with a TEV site insertion following residue K71 in Enc exhibited the highest overexpression and HBCM2 release efficiencies compared to other variants but were deficient in cage formation. HBCM2 was purified from the highest expressing variant following TEV protease digestion and was found to be highly active in inhibiting E. coli growth (MIC = 5 μg/ml). Our study demonstrates the potential use of the Enc system to enhance expression of AMPs for biomanufacturing and therapeutic applications.     

Identifying kinetically stable proteins with capillary electrophoresis

Unlike most proteins, which are in equilibrium with partially and globally unfolded conformations, kinetically stable proteins (KSPs) are trapped in their native conformations and are often resistant to harsh environment. Based on a previous correlation between kinetic stability (KS) and a protein's resistance to sodium dodecyl sulfate (SDS), we show here a simple method to identify KSPs by SDS‐capillary electrophoresis (CE). Control non‐KSPs were fully denatured by SDS and formed protein:SDS complexes that exhibited similar mobility in CE. In contrast, KSPs bound fewer SDS molecules, and showed a very different migration time and peak pattern in CE, thereby providing some insight about the structural heterogeneity of SDS:protein complexes and the relative KS of the various proteins.     

Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis

Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (maximum excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chemicals can be carried out prior to, during, or after separations. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the analysis of polymerase chain reaction products, DNA sequencing, trace analysis of proteins, and single cell analysis have been presented.     

Analytical and micropreparative capillary electrophoresis of the peptides from calcitonin.

The capillary electrophoresis (CE) of peptide fragments from the tryptic digest of salmon calcitonin and elcatonin has been carried out in H2O- and D2O-based buffer solutions. Analysis in heavy water was found to be superior to that in H2O especially at a pH or pD of 7.93. From a single CE run on elcatonin digest we were also able to harvest three pure cleavage peptides in sufficient quantity to determine each amino acid residue by protein sequencing. The order of elution from CE agreed with that predicted on the basis of net charge calculated for each peptide.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.     

Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing

Strategies reported for the separation of proteins in capillary zone electrophoresis and capillary isoelectric focusing are reviewed. The strategies are grouped into two categories: coated capillaries and buffer/sample additives. Success attained with each case and also, more importantly, the limitations of the methodology are discussed. Recent results from our own laboratory in the area of capillary isoelectric focusing in uncoated, fused silica capillaries using additives are summarized. The advantages and disadvantages of coated columns vs. additives are delineated.     

A semiempirical model for the electrophoretic mobilities of peptides in free-solution capillary electrophoresis

In this study an attempt is made to explore the effect of a peptide's size, charge, and hydrophobicity on its electrophoretic mobility (mu) as measured by free-solution capillary electrophoresis with the aim of developing a semiempirical model which incorporates these effects. The effects of peptide size (which is measured by the number of amino acids in the polypeptide chain (n] and charge on mu are independently determined by experiment in a single solvent system and combined to give the relationship (formula; see text) where the constant 5.23 X 10(-4) is postulated to depend on the solvent system used. The form of Eq. [A.1] was confirmed, and the values of the constants 5.23 X 10(-4) and 2.47 X 10(-5) were determined, by measuring the electrophoretic mobilities of 40 peptides varying in size from 3 to 39 amino acids and varying in charge from 0.33 to 14.0. Furthermore, the effect of noncharged neutral amino acids on mobility was investigated and shown to be present, but only as a minor perturbation on the effects of size and charge.     

尿样中三种蛋白质的毛细管电泳分离检测方法研究

目的:建立毛细管电泳分离测定人尿样中转铁蛋白、白蛋白和血红蛋白的新方法.方法:通过选择运行缓冲溶液种类及浓度、pH、表面活性荆种类及浓度、分离电压、进样时间对蛋白质分离效果的影响,优化了毛细管电泳法分离转铁蛋白、白蛋白和血红蛋白的条件.结果:利用此方法测定三种蛋白质的含量,浓度在0.01到1.00 g L-1范围内与峰电流呈良好的线性关系,检出限均为10-4 g L-1.结论:所建立的方法用于人尿样中转铁蛋白、白蛋白和血红蛋白的测定,结果满意.     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.