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.     

On the nature of the forces controlling selectivity in the high performance capillary electrochromatographic separation of peptides

In this minireview, the nature of the forces controlling selectivity in the high performance capillary electrochromatographic (HP-CEC) separation of peptides has been examined. For uncharged and charged peptides, a synergistic interplay occurs in HP-CEC systems between adsorptive/partitioning events and electrokinetically driven motion. Moreover, at high field strengths, both bulk electrophoretic migration and surface electrodiffusion occur. Thus, the migration behavior of peptides in different HP-CEC systems can be rationalized in terms of the combined consequences of these various processes. Moreover, in HP-CEC, the buffer electrolyte interacts with both the peptide analytes and the sorbent as bulk phenomena. These buffer-mediated processes control the solvational characteristics, ionization status and conformational behavior of the peptides as well as regulate the double-layer properties of the sorbent, and the ion flux and electro-osmotic flow characteristics of the HP-CEC system per se. These buffer electrolyte effects mediate mutual interactions between the peptide and the sorbent, irrespective of whether the interaction occurs at the surface of microparticles packed into a capillary, at the surface of a contiguous monolithic structure formed or inserted within the capillary or at the walls of the capillary as is the case with open tubular HP-CEC. Diverse molecular and submolecular forces thus coalesce to provide the basis for the different experimental modes under which HP-CEC can be carried out. As a consequence of this interplay, experimental parameters governing the separation of peptides in HP-CEC can be varied over a wide range of conditions, ensuring numerous options for enhanced selectivity, speed, and resolution of peptides. The focus of the peptide separation examples presented in this minireview has been deliberately restricted to the use of HP-CEC capillaries packed with n-alkyl-bonded silicas or mixed-mode strong ion exchange sorbents, although other types of sorbent chemistries can be employed. From these examples, several conclusions have been drawn related to the use of HP-CEC in the peptide sciences. These observations confirm that variation of a specific parameter, such as the pH or the content of the organic solvent modifier in the buffer electrolyte, simultaneously influences all other physicochemical aspects of the specific HP-CEC separation. Peptide selectivity in HP-CEC thus cannot be fine-tuned solely through the use of single parameter optimization methods. In this context, HP-CEC differs significantly from the analogous reverse phase high performance liquid chromatography (RP-HPLC) procedures with peptides. Rather, more sophisticated multiparameter optimization procedures, involving knowledge of (a) the field strength polarity, (b) its contour and flux characteristics, (c) effects of buffer electrolyte composition and pH, (e) the influence of the temperature, and (f) the impact of the sorbent characteristics, are required if the full capabilities offered by HP-CEC procedures are to be exploited. In this minireview, the HP-CEC migration behavior of several different sets of synthetic peptides has been examined, and general guidelines elaborated from these fundamental considerations to facilitate the interpretation and modulation of peptide selectivity in HP-CEC.     

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.     

Detection of a genetic variant, lysine→glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330–446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine→glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.     

Using affinity capillary electrophoresis to study the interaction of the extracellular binding domain of erythropoietin receptor with peptides.

We have shown that affinity capillary electrophoresis (ACE) can be utilized to screen peptides that bind to the extracellular binding domain of the erythropoietin receptor (EBP). The comparison of the cyclic peptides GGTYSCHFGPLTWVCKPQGG (EMP1) GGTYSCHFGPLTAVCKPQGG (EMP13), and LGRKYSCHFGPLTWVCQPAKKD (EMP37) with the linear peptides HFGPLTWV (EMP26) and FMRF as ACE buffer additives were investigated. When EMP1 and EMP37 were the buffer additives, an abrupt change in the electrophoretic mobility of EBP was observed in the electropherogram. When EMP13, EMP26, and FMRF were examined under identical ACE conditions as EMP1 and EMP37, no significant change in the electrophoretic mobility of EBP was observed. These results correlate well with previously reported IC50 competitive binding data; that is, EMP1 and EMP37 bind to EBP while EMP13 and EMP26 bind very weakly. These observations strongly infer that peptide.EBP dimerization were induced by EMP1, and EMP37 but not by EMP13, EMP26 or FMRF. This ACE method provides a rapid tool for the detection of small peptides or drugs that bind to EBP.     

High-performance separation methods in the analysis of a new peptide family: the galanins

An analytical investigation of a new peptide family, the human galanins and their fragments, was carried out by reversed-phase HPLC, capillary zone electrophoresis (CZE) at different pH values and micellar electrokinetic capillary chromatography (MECC) in phosphate-borate-sodium dodecyl sulphate buffer. None of the methods seems to be superior to the others. The complementary nature of the electrophoretic methods is obvious when the profiles of peptides are compared; impurities not separated by HPLC are separated by CZE or MECC and vice versa. With these three different separation methods, a more complex analytical control of the synthetic work can be achieved.     

Electrokinetic studies of inorganic coated capillaries

The procedures for the preparation of silica capillaries coated with titanium oxide or aluminum oxide are developed. These inorganic coated capillaries are studied for their applicability in capillary electrophoresis. The points of zero charge are measured as pH 5 and pH 7 for titanium oxide- and aluminum oxide-coated capillaries, respectively. Both titanium oxide and aluminum oxide coatings give better protein separations in comparison to the use of fused-silica capillaries. Separation efficiency of lysozyme as model protein is measured in the range of 20 000 theoretical plates/m of inorganic coated capillaries. However, the hydrophobic interaction between proteins and modified capillary wall possibly contributes to the tailing of observed protein peaks.     

Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation

Capillary electrophoresis using SDS in phosphate buffer provides high resolution and short separation time for peptide and protein hydrolysate amino acids after derivatization with phenylisothiocyanate. The phenylthiocarbamyl derivatives are quantified in the picomole and femtomole range at signal-to-noise ratios better than 3:1 (for 50 fmol) and with a linearity correlation coefficient averaging 0.9938. The migration time and peak area variabilities were on average 1.1 and 2.7%, respectively. Complete separation of all the 18 amino acids normally found in polypeptide hydrolysates is achieved in less than 30 min using 75-microm capillaries while 50-microm capillaries require less than 15 min. Analysis of peptide and protein hydrolysates in the range 10-600 residues revealed excellent agreement with the known compositions at sensitivities better by large factors than the corresponding HPLC methodology (about 20-fold) and conventional ninhydrin-based analysis (about 1000-fold).     

Electromigration behavior of poly-(L-glutamate) conformers in concentrated polyacrylamide gels

During electrophoretic separation of anionic polyamino acids, resolution according to the number of peptide units can be achieved in capillaries filled with hydrophilic gels. While polyaspartate preparations yield single peaks for the individual oligomers at pH above 8.0, polyglutamates exhibit an anomalous behavior of peak splitting, which is attributed here to the separation of the oligopeptide conformers. An Asp-Glu (1:1) copolymer yields single peaks under similar conditions. At pH near 4.5, where polyglutamate is expected to exist in its α-helix form, peak splitting disappears. Upon heating to 95°C for at least 120 h (procedure described to transform the α-helix into a β form), peak splitting disappeared, but could be reestablished after cooling for several days. When a highly charged cation spermine was added to the operational electrolyte, triple peaks appeared in the electropherogram due to the ion-pair formation. The largest peak in every triplet has been tentatively assigned to the α-helix form. The electrophoretic results described have been largely supported by CD spectra. © 1993 John Wiley & Sons, Inc.     

连钱草药材的高效毛细管电泳指纹图谱研究

建立了不同产地连钱草药材的高效毛细管电泳(HPCE)指纹图谱,并进行了比较。连钱草药材采用体积分数75%乙醇蒸馏提取;采用毛细管区带电泳法制定指纹图谱,电泳条件如下:未涂层融熔石英毛细管50μm I.D.×375μm O.D.(总长62 cm,有效长度53.5 cm),电泳缓冲液为含体积分数15%乙腈的50 mmol.L-1硼酸缓冲液(pH9.2),检测波长278 nm,操作电压25 kV,柱温25℃;以对乙酰氨基酚为参照物,建立了连钱草药材的数据化指纹图谱(相对迁移时间和相对峰面积)。结果表明:5种连钱草药材(4个产地和对照药材)具有16个共有指纹峰,不同产地连钱草药材指纹图谱的峰重叠率都低于60.3%,共有峰相对峰面积及8强峰均差异较大;连钱草药材的主要化学成分种类和质量分数均因产地不同而有差异;HPCE指纹图谱法具有较好的稳定性、精密度和重现性,简便,快速,可作为连钱草药材的质量控制方法。     

Determination of urinary succinylacetone by capillary electrophoresis for the diagnosis of tyrosinemia type I

The presence of succinylacetone in urine or blood or amniotic fluid is pathognomonic of an inherited metabolic disorder, named tyrosinemia type I. We developed a capillary electrophoretic method for the fast analysis of succinylacetone in urine samples. The separation was performed at reversed polarity mode using either a cationic surfactant as the buffer additive, or a capillary coated with a positively charged polyelectrolyte. Under these conditions, urine samples were directly injected to the capillary without any pretreatment step. The utility of the method was demonstrated by the identification of succinyacetone in urine from patients with hereditary tyrosinemia type I. For all patients, diagnostic peaks at the expected migration times were detected. The developed method is rapid, simple, inexpensive, and suitable for the determination of succinylacetone in clinical urine samples.     

Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay

A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions.     

Towards a quantitative free flow electrophoresis and its application to particle size separations

The possibility to quantify free-flow electrophoresis (FFE) data was explored in application to 6 negatively charged polystyrene size standards in the size range of 73 to 762 nm diameter. Peak fraction numbers in FFE were shown to be proportional to mobilities of the particles, determined by capillary zone electrophoresis in the identical buffer. Standard deviations of peak fraction numbers demonstrate a high degree of intra-experimental reproducibility while inter-experimentally, a variability of 1 to 5 peak fraction numbers within 28 fractions was found. A relative mobility (Rf) scale for peak identification in FFE based on the free mobility of the dye, SPADNS, allowed for the utilization of the entire electrophoretic migration path but failed to improve the precision of fraction numbers in view of the substantial zone spreading of the dye. Mobility differences between particles increased upon lowering the ionic strength of the electrophoretic buffer. Peak width increased with particle size in inverse relation to ionic strength.     

The CEC behaviour of several synthetic peptides related to the activin betaA-betaD subunits.

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme.

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.     

Rapid screening of monoamine oxidase B inhibitors in natural extracts by capillary electrophoresis after enzymatic reaction at capillary inlet

A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N′-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH = 7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K_i, K_m and IC₅₀ based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD = 1.8%) and peak area (RSD = 3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.     

Genetic variability of leaf esterases in Triticum aestivum L. 2n=6x=42

Summary Starch gel electrophoresis with two different buffer systems and several substrates and inhibitors have been used to study the electrophoretic variability of esterases in leaves of cultivars of Triticum aestivum. Each one of the buffer systems showed different levels of variability, according to the electrophoretic patterns. At the same time green and etiolated leaves showed different patterns in each buffer system. The variability was dependent upon the developmental stage of the leaves. According to the results from chromosomal location, the genes controlling esterases in green leaves were located in homoeology group 3, while the genes controlling esterases in etiolated leaves were in homoeology group 6. But both esterase isozymes showed a similar electrophoretic migration and a similar reponse to substrates and inhibitors. The possible origin of both sets of genes due to an interchromosomal duplication is discussed.     

Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2)

Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK) is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32)P [counts per minute (CPM)] for each peptide substrate was determined by the radiolabel assay using [γ-(32)P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135) were phosphorylated by other enzymes (PKA, PKCα, and ERK1), R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.     

Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference

The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.