Application of capillary isotachophoresis in peptide analysis

This paper gives a broad and detailed review of the applications of one of the modern high-performance electromigration separation techniques--capillary isotachophoresis (ITP)--in peptide analysis. Examples are presented of the utilization of capillary ITP for peptide analysis in the fields of chemistry, general and clinical biochemistry, biology, biotechnology, pharmacy and the food industry. The complete composition of all the electrolyte systems used for peptide ITP analyses in both cationic and anionic techniques is given in tabular form. According to the purpose of analysis the applications are divided into several sections: model studies, determination of physico-chemical characteristics, purity control of both intermediate and final peptide preparations, including the determination of low-molecular-mass ionogenic admixtures, and the analysis of peptides in biological fluids and tissue extracts. In addition to the main applications the theoretical and methodological aspects of peptide ITP analysis are discussed. The basic electromigration properties of peptides (their polyampholyte character, effective and absolute mobilities, acid-base equilibria) are explained and the selection of parameters for peptide ITP analysis is described in detail. The advantages and disadvantages of ITP compared with other electrophoretic and chromatographic methods used for peptide analysis are discussed.     

Improving the detection limit in capillary isotachophoresis using asymmetric neutralisation reaction boundary

An online method involving transient electrokinetic dosing and ITP with neutralization reaction boundary (NRB) and/or carrier ampholyte-free isoelectric focusing (CAF IEF) was developed for the preconcentration, preseparation, and analytical determination of glyphosate in aqueous samples containing low concentrations of the analyte of interest. Various parameters were investigated in the framework of an optimization study with the aim of achieving the maximum concentration limit of detection (cLOD) decrease in minimum time. The proposed method used CAF IEF and/or ITP with NRB. The sample was dosed to the column on the stationary reaction boundary (CAF IEF) and/or moving reaction boundary (ITP with NRB), whereat a sharp pH step exists. Here, charge reversal was due to the ampholytes, and/or acid accumulation occurred because of charge loss. Similarly, the accumulated sample was mobilized with TE and analyzed using classical ITP in the second analytical column. Glyphosate (GLY), the analyte of interest, was chosen as a model substance for ITP with NRB and preconcentration as well as focusing preconcentration and CAF IEF using the asymmetric purpose-built NRB. On one side of the asymmetric boundary was the zone of acidic pH; while the opposite side comprised a neutral/basic non-conductive zone of the ampholyte—in this case, GLY. Such an arrangement enables the use of a lower pH on the acidic side, which allows the focusing of strongly acidic ampholytes and the accumulation of weak acids. The electrolyte composition and the dosing time were optimized, and a 14-fold accumulation was achieved in 25 min compared to that by classical ITP and a 180-fold accumulation was achieved through CAF IEF and preconcentration with a glyphosate sample. Both methods are simple and can be conducted using all commercial ITP systems.     

A Study of peptide-peptide interaction by matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study peptide-peptide interaction. The interaction was seen when 6-aza-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a more acidic matrix, alpha-cyano-4-hydroxycinnamic acid (pH 2.0). In the present study, we show that dynorphin, an opioid peptide, and five of its fragments that contain two adjacent basic residues (Arg6-Arg7), all interact noncovalently with peptides that contain two to five adjacent acidic residues (Asp or Glu). Two other nonrelated peptides containing two (Arg6-Arg7) or three (Arg1-Lys2-Arg3) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjacent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin only cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin fragments (containing the motif RR), and an acidic peptide (minigastrin), the acidic peptide preferentially complexed with dynorphin. If the concentration of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absence of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and concentration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex formation, and (3) the complex did not form with fragments of dynorphin or unrelated peptides that did not contain the motifs RR or RKR, nor with a fragment of dynorphin where Arg7 was mutated to a phenylalanine residue. These findings strongly suggest that peptide-peptide interaction does occur, and can be studied by MALDI if near physiologic pH is maintained.     

Dynamic pH junction technique for on-line preconcentration of peptides in capillary electrophoresis

A method based on the presence of a dynamic pH junction within the capillary to induce band narrowing for enhanced detection sensitivity for some peptides is presented. This technique is predicated on a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the basic BGS zone. Larger-than-usual injection volumes of samples in relatively high-conductivity matrices were enabled, without degrading peak shape, resolution and efficiency. The size of the original sample plug was reduced by as much as 38-fold, and improvement in detector response in terms of peak height by as much as 124-fold was obtained. The effects of pH and concentration of the sample matrix, and the length of sample injection on the efficiency of the technique are discussed.     

Base-induced transient isotachophoretic stacking of acidic drugs in capillary zone electrophoresis

Online sample concentration of acidic drugs by transient isotachophoresis (t-ITP) with the injection of a base is described in capillary zone electrophoresis (CZE). A positively coated capillary was conditioned with background electrolyte (ammonium acetate at pH 6). A long plug of sample solution (S) prepared in ammonium acetate was then hydrodynamically injected followed by the base (tetrapropylammonium hydroxide). A negative voltage was applied and caused the hydroxide ions from the base to penetrate the S zone and created a pH junction that swept through the S zone. The analytes stack at the junction where the mechanism of focusing was transient ITP with the acetate and hydroxide ions as leading and terminating ions, respectively. The concentrated analytes separated in co-EOF CZE once the hydroxide was exhausted. The base stacking strategy was tested using hypolipidemic, nonsteroidal anti-inflammatory, and diuretic drugs, and afforded 19-37 improvements in peak height.     

Direct determination of celiprolol in human urine using on-line coupled ITP-CZE method with fiber-based DAD

The present work illustrated possibilities of column-coupling electrophoresis combined with DAD for the direct quantitative determination of trace drug (celiprolol, CEL) in clinical human urine samples. ITP, on-line coupled with CZE, served as an ideal injection technique (high sample load capacity, narrow and sharp drug zone). Moreover, the ITP provided an effective on-line sample pretreatment (preseparation, purification and preconcentration of the drug) producing analyte zone suitable for its direct detection and quantitation in CZE stage. Spectral DAD in comparison with single wavelength ultraviolet detection enhanced value of analytical information (i) verifying purity (i.e., spectral homogeneity) of drug zone (according to differences in spectrum profiles when compared tested and reference drug spectra) and (ii) indicating zones/peaks with spectra similar to the drug spectrum (potential structurally related metabolites). The characterization of trace analyte signals superposed on the baseline noise was more definite thanks to the application of background correction and smoothing procedure to the raw DAD spectra (producing relevant spectral response). The proposed ITP-CZE-DAD method was characterized by favorable performance parameters for CEL in urine matrices {e.g., the lower limit of quantification was 9.7 ng/mL, RSD and relative error of the determinations were lower than 3% (precision) and 1% (accuracy), respectively, analyte peak exhibited spectral homogeneity (reflecting separation selectivity), separation efficiency was 84,500 theoretical plates} and successfully applied in a trial pharmacokinetic study of CEL.     

Computer-assisted choice of discrete spacers for anionic isotachophoresis separations

In isotachophoresis (ITP), the sample constituents migrate, depending on their concentrations in the loaded sample, either in fully developed zones or in the boundary layers between the zones of constituents of the corresponding effective mobilities. The latter (spike) migration mode is analytically beneficial in selective detections of trace analytes, especially, when appropriately chosen discrete spacers minimize detection interferences due to matrix constituents. To facilitate a search for suitable mixtures of discrete spacers, a two-step calculation procedure was developed in this work. Using a pool of discrete spacers consisting of 42 anionic and zwitterionic constituents, this procedure was shown effective in the anionic ITP separations performed at pH = 6.5-10.0. Besides the predictions of the migration orders, it was helpful in identifying the spacing constituents that could cause resolution problems due to an uncertainty with which pH of the leading electrolyte solution is known. The ionic mobility and pKa data, taken for the spacing constituents from the literature and the ones obtained from the ITP experiments carried out in this work, were used in the calculations performed in a context with the choice of spacers. Although the data obtained from the ITP experiments provided better results, small uncertainties with which they were acquired (attributable to fluctuations in the experimental conditions) set practical limits in the calculation based choice of multi-component mixtures of the spacing constituents.     

Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis

An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.     

Window optimization in isotachophoresis superimposed on capillary zone electrophoresis

A sample stacking procedure to which a specific combination of electrolyte solutions is applied is isotachophoresis (ITP) superimposed on capillary zone electrophoresis (CZE), a so-called ITP/CZE system. In ITP/CZE some components migrate in an ITP fashion on top of a background electrolyte, and the other analytes migrate in a zone electrophoretic manner. For such a system, the leading electrolyte consists of a mixture of an ionic species, L1, of high mobility (the leading ion of the ITP system), an ionic species, L2, of low mobility (the coions of the CZE system), and a buffering counter-ionic species, whereas the terminating solution only contains the ionic species L2 and the buffering counterions. The zones of the components migrating in the ITP/CZE mode are sharp owing to the self-correcting properties of the zones and the concentrations of the L1 ions of the system. Mobility windows can be calculated, indicating which ions can migrate in the ITP/CZE mode. In this article mobility windows are calculated by applying both strong and weak acids as L1 and L2 ions and it appears that mobility windows can be optimized by chosing different ratios of L1 and L2 as well as different pH values. It is possible to construct very narrow mobility windows, and thereby choose which component of a sample solution can be concentrated, and to what concentration, in a very selective way. The big advantage of ITP/CZE compared with applications such as transient ITP and transient stacking is that the stacked sample ionic species migrate in the ITP mode during the whole experiment; furthermore, they do not destack. Experimentally obtained electropherograms validate the calculated mobility windows for the ITP/CZE mode.     

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers). Acidity of phosphinic acid moiety in peptides with basic amino acid residues was determined from the dependence of effective mobility of these peptides on pH in the acid pH region (pH 1.4-2.8). Additionally, separation of diastereomers of some peptides was achieved.     

Contribution of acidic amino residues to the adsorption of peptides onto a stainless steel surface

The role of the acidic amino acid residues in the adsorption of peptides/proteins onto stainless steel particles was investigated using a peptide fragment from bovine beta-lactoglobulin, Thr-Pro-Glu-Val-Asp-Asp-Glu-Ala-Leu-Glu-Lys (T5 peptide), which has a high affinity to a stainless steel surface at acidic pHs, and its mutant peptides substituted with different numbers of acidic amino acid residues. The adsorption behavior of the mutant peptides as well as the T5 peptide were studied at pH 3 with respect to concentration and ionic strength dependencies and the reversibility of the adsorption process. The behavior of the peptides was generally characterized as two distinct irreversible adsorption modes, Mode I and Mode II. In Mode I, the amounts adsorbed lay on the ordinate at zero equilibrium concentration in the solution, while in Mode II, the amount adsorbed increased with increased equilibrium concentration. The area occupied by the peptides was predicted by molecular mechanics and molecular dynamics. The state of the peptides, when adsorbed, was investigated using FT-IR analysis. The FT-IR analyses revealed that the side carboxylic groups of the peptides adsorbed on the stainless steel surface were ionized, while they were unionized in the solution at pH 3. Thus, the interactions between the carboxylic groups of the peptide and the stainless steel surface can be considered to be largely electrostatic. The peptide having four acidic amino acid residues took a maximum adsorbed amount, the reason for which is discussed.     

Separation and investigation of structure-mobility relationships of insect oostatic peptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been applied to qualitative analysis, separation, and physicochemical characterization of synthetic insect oostatic peptides (IOPs) and their derivatives and fragments. Series of homologous IOPs were separated in three acidic background electrolytes (BGEs; pH 2.25, 2.30, 2.40) and an alkaline BGE (pH 8.1). Best separation was achieved in acid BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The effective electrophoretic mobilities, mu(ep), of all IOPs in four BGEs were determined and several semiempirical models correlating effective mobility with charge-to-size ratio (mu(ep) versus q/Mr k) were tested to describe the migration behavior of IOP in CZE. None of models was found to be unambiguously applicable for the whole set of 20 IOPs differing in size (dipeptide - decapeptide) and charge (-2 to +0.77 elementary charges). However, a high coefficient of correlation, 0.9993, was found for the subset of homologous series of IOPs with decreasing number of proline residues at C-terminus, H-Tyr-Asp-Pro-Ala-Prox-OH, x = 6 - 0, for the dependence of mu(ep) on q/Mr k with k = 0.5 for IOPs as anions in alkaline BGE and with k = 2/3 for IOPs as cations in optimized acidic Tris-phosphate BGE. From these dependences the probable structure of IOPs in solution could be predicted.     

Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography

This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange. identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2'-dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol (pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species between the two samples. Peptides obtained from proteins that were up-regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted.     

Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two-layer sample preparation

While surfactants are commonly used in preparing protein samples, their presence in a protein sample can potentially affect the enzymatic digestion process and the subsequent analysis of the resulting peptides by mass spectrometry. The extent of the tolerance of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to surfactant interference in peptide analysis is very much dependent on the matrix/sample preparation method. In this work the effects of four commonly used surfactants, namely n-octyl glucoside (OG), Triton X-100 (TX-100), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS), for biological sample preparation on trypsin digestion and MALDI-MS of the resulting digest are examined in detail within the context of using a two-layer method for MALDI matrix/sample preparation. Non-ionic and mild surfactants, such as OG, TX-100 or CHAPS, are found to have no significant effect on trypsin digestion with surfactant concentrations up to 1%. However, TX-100 and CHAPS interfere with the subsequent peptide analysis by MALDI-MS and should be removed prior to peptide analysis. OG is an MS-friendly surfactant and no effect is observed for MALDI peptide analysis. The effect of SDS on trypsin digestion in terms of the number of peptides generated and the overall protein sequence coverage by these peptides is found to be protein dependent. The use of SDS to solubilize hydrophobic membrane proteins, followed by trypsin digestion in the presence of 0.1% SDS, results in a peptide mixture that can be analyzed directly by MALDI-MS. These peptides are shown to provide better sequence coverage compared with those obtained without the use of SDS in the case of bacteriorhodopsin, a very hydrophobic transmembrane protein. This work illustrates that MALDI-MS with the two-layer sample preparation method can be used for direct analysis of protein digests with no or minimum sample cleanup after proteins are digested in a solution containing surfactants.     

Analysis of amino acids by combination of carrier ampholyte-free IEF with ITP

The use of carrier ampholyte-free IEF (CAF-IEF) with ITP mobilization and conductivity detection in ITP mode for preconcentration and analysis of amino acids is demonstrated. The analytical procedure consists of three subsequent steps. In the first step, amino acids are continuously dosed from an infinite volume reservoir by electromigration to the column, where a sharp, stationary neutralization reaction boundary (NRB) is created in between acidic and basic primary electrolyte. Here, amino acids are selectively focused (trapped), if their pI falls to the pH difference on both sides of the NRB (pH gap). Amino acids create sharp rectangular zones, arranged according to their pI values. In the second step, focused zones are mobilized. After accumulation of the detectable amount of amino acids, dosing electrolyte in the infinite volume reservoir is changed for the mobilizing electrolyte. The migration mode is changed from CAF-IEF to ITP and substances start to migrate toward the analytical capillary. In the third step, analytes are transferred into the analytical column equipped with a conductivity detector and are detected in the new leading electrolyte in an ITP migration mode. The presented CAF-IEF-ITP-ITP with time-dependent accumulation of the large-volume sample enables to achieve in a reasonable time a 100 times lower c-LOD (here in orders of nmol/L), than can be reached by conventional hyphenated ITP-ITP.     

Combination of large volume sample stacking and dynamic pH junction for on-line preconcentration of weak electrolytes by capillary electrophoresis in comparison with isotachophoretic techniques

An on-line preconcentration capillary electrophoresis (CE) technique, which combines a large volume sample stacking with a dynamic pH junction technique, is introduced in this paper. This dynamic pH junction with co-electroosmotic migration is formed between sodium borate pH 9.5 and sodium phosphate pH 2.5 with 150 mM sodium dodecylsulfate (SDS). A full capillary based injection allows determination of weak acidic compounds at ppb concentration levels (achieved LOD for benzoic acid was 11 nmol L(-1)). The proposed preconcentration method was compared with ITP/ITP (LOD 120 nmol L(-1)), ITP/CZE (LOD 740 nmol L(-1)) and a simple CZE method (LOD 23,330 nmol L(-1)). The analytical potential of this method was assessed with juice test samples.     

Solvent-free MALDI-MS for the analysis of biological samples via a mini-ball mill approach

An efficient, low sample load mini-ball mill (MBM) sample preparation procedure was developed for solvent-free MALDI analysis of peptides and proteins. Picomole sample amounts can be handled conveniently, with 30 s grinding times being sufficient. Matrix purity and molar analyte/matrix ratios are not as critical as with methods employing solvent. Ammonium salt is employed for protonation of the peptide and suppression of sodiation. This strategy allows for peptide mapping and other biochemical manipulations to be performed prior to MBM sample preparation and mass analysis. The analysis of bovine serum albumin (66 kDa) yielded good results, indicating that higher molecular weight proteins are accessible. A semi-solvent-free strategy by the MBM sample preparation method is also described.     

Effects of the sample matrix on the separation of peptides by high performance capillary electrophoresis

The effects of the sample matrix on the separation of peptides by HPCE has been investigated. Under both acidic and alkaline conditions, use of 25–30 mM salts in the sample zone resulted in much better resolution than did 100 mM salts. Prefocusing effects and acceleration of elution were also observed. These results agree well with the theory developed by Everaerts. In this paper a practical guide for high sensitivity, high resolution separation of salt-containing peptide mixtures is proposed.     

Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins

Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.