Transient isotachophoresis in carrier ampholyte-based capillary electrophoresis for protein analysis

Transient ITP (t-ITP) has been used in carrier ampholyte-based CE (CABCE) to enhance the sensitivity of protein analysis. The characteristics of carrier ampholytes (CAs) narrow pH cuts-based buffers, when used as BGEs in CE are compatible with t-ITP requirements. Indeed, being the sole buffering species of such solutions, CAs impose a pH close to their pI. Thus, in these solutions, the CAs possess low electrophoretic mobility. As a consequence, by adding an ionic component with high electrophoretic mobility either in the studied sample or in the BGE, a t-ITP step can be generated. This has first been demonstrated for protein test mixtures. Then, the combination of t-ITP with CABCE has been applied to study a real sample, the bovine milk.     

Protein tryptic digests analyzed by carrier ampholyte-based capillary electrophoresis coupled to ESI-MS

In this study, narrow pH cuts of carrier ampholytes have been used as buffers in CE for the analysis of protein tryptic digests. Their low conductivity allows very efficient separations under high electric field strength without inducing any significant Joule heating. In this study, the capabilities of narrow pH cuts of carrier ampholytes for the separation of protein tryptic digests have been assessed. Three proteins of different molecular masses have been studied: cytochrome C (horse heart), beta-lactoglobulin B (bovine) and human transferrin. Efficient, rapid and repeatable separations of the peptides resulting from the tryptic digestion have been achieved in this buffer. Moreover, the feasibility of the coupling of carrier ampholyte-based capillary electrophoresis with ESI-MS has been demonstrated through the study of the cytochrome C tryptic digest.     

Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.     

Loading capacity of carrier ampholytes--based buffers in capillary electrophoresis

Narrow pH cuts of carrier ampholytes (CAs), originally designed for IEF, have been used as BGEs in CE. Their physicochemical properties, rather high buffering capacity and low conductivity, allow very efficient protein separations under high electric field strength. Due to their isoelectric properties, CA BGEs are expected to present a low ionic concentration and consequently a low loading capacity. In this study, we developed a simple method that allows the estimation of the loading capacity of a UV-absorbing BGE by CE. We first characterized in terms of loading capacity, classical ammediol-chromate UV-absorbing BGEs and a 10 mM histidine solution, a classical isoelectric buffer. Then, the loading capacity of four different CA-based BGEs has been assessed. Experimental results have shown that the CA-based buffers were presenting a rather high loading capacity, comparable to classical buffer ones and far higher than the one of the 10 mM histidine solution.     

Determination of pK(a) values of diastereomers of phosphinic pseudopeptides by CZE

A CE method was used for the determination of acidity constants (pK(a)) of a series of ten phosphinic pseudopeptides, which varied in number and type of ionogenic groups. Effective electrophoretic mobilities were measured in the 1.8-12.0 pH range in the BGEs of constant ionic strength of 25 mM. Effective electrophoretic mobilities, corrected to standard temperature of 25 degrees C, were subjected to non-linear regression analysis and the obtained apparent pK(a) values were recalculated to thermodynamic pK(a)'s by extrapolation to zero ionic strength according to the extended Debye-Hückel model. The pK(a) values of the phosphinic acid group fell typically in the 1.5-2.25 interval, C-terminal carboxylic groups in the 2.94-3.50 interval, carboxylic groups of the lateral chain of glutamate and aspartate in the 4.68-4.97 interval, imidazolyl moiety of histidine in the 6.55-8.32 interval, N-terminal amino groups in the 7.65-8.28 interval and epsilon-amino group of the lateral chain of lysine in the 10.46-10.61 interval. Further, separation of diastereomers of the phosphinic pseudopeptides was investigated in achiral BGEs. Evaluation of the resolution of the diastereomers as a function of pH of the BGE revealed that most suitable pH region for separation of the diastereomers is around the pK(a) values of the central phosphinic acid group of the pseudopeptides. Successful separation of some diastereomers was, however, achieved in the neutral and alkaline BGEs as well.     

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.     

Separation of diastereomers of phosphinic pseudopeptides by capillary zone electrophoresis and reverse phase high‐performance liquid chromatography

Capillary zone electrophoresis (CZE) and reverse phase high‐performance liquid chromatography (RP‐HPLC) were used for separation of diastereomers of phosphinic pseudopeptides in achiral separation media. A set of phosphinic pseudopeptides, i. e. peptides with one peptide bond substituted by phosphinic acid moiety ‐PO₂^–‐CH₂‐ derived from the structure N‐Ac‐Val‐AlaB(‐CH₂)Leu‐His‐NH₂ synthesized as a mixture of four diastereomers was used. Separations of diastereomers by CZE were carried out in Tris‐phosphate background electrolytes in the pH range 1.1–3.2 and at least partial separation of the four diastereomers of each pseudopeptide was achieved. A routinely used RP‐HPLC method (C₁₈‐silica column and water/acetonitrile/trifluoroacetic acid mobile phase) was also capable of resolving the diastereomers. In addition, since individual diastereomers of majority of the pseudopeptides were isolated by RP‐HPLC it was possible to check the purity of these RP‐HPLC separated diastereomers and to compare the migration order of the diastereomers in CZE with their elution order in RP‐HPLC. The results obtained by CZE and RP‐HPLC demonstrate a complementarity of both methods in analysis and separation of phosphinic pseudopeptides including their diastereomers.     

Capillary isoelectric focusing-mass spectrometry for shotgun approach in proteomics

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.     

Autofocusing and ESI-MS analysis of protein digests in a miniaturized multicompartment electrolyzer

Free-solution IEF of protein digests was studied in a newly introduced MicroRotofortrade mark multicompartment electrolyzer. The fractionation was performed in a cylindrical separation chamber divided into ten compartments with or without the addition of carrier ampholytes. In the case of autofocusing mode of operation, the tryptic digest itself served as the mixture of ampholytes leading to the separation of the peptides with well-defined pI's. The focusing process was monitored visually using colored pI markers. The resulting fractions from both modes of the separation were analyzed by CE and electrospray-TOF mass spectrometer using electrospray tips microfabricated in polyimide. Additional experiments, aiming at visualization of the mass flux within the focusing compartments were performed using isotachophoretic migration of color cationic tracers. The study considered the autofocusing of both the peptides with well-defined narrow pI's as well as those showing negligible net charge in a broader pH range. Although not all peptides in the protein digests have well-defined pI's the autofocusing process can preseparate many of them leading to higher S/N in the ESI-MS signals and improved protein sequence coverage.     

The free solution electrophoretic mobility of peptides by a bead modeling methodology

A bead modeling methodology, BMM, discussed previously to compute the free solution electrophoretic mobility of peptides [H. Pei, Y. Xin, S.A. Allison, J. Sep. Sci. 31 (2008) 554-564], is generalized to avoid the approximation of orientationally preaveraging hydrodynamic interaction. In general, peptide mobilities computed without preaveraging are lower by about 2%. The BMM is then used to study the free solution electrophoretic mobility of several insect oostatic peptides reported previously in a variety of different buffer systems ranging in pH from 2.25 to 8.1 [V. Solinova, V. Kasicka, D. Koval, J. Hlavacek, Electrophoresis, 25 (2004) 2299-2308]. With minor adjustment of the intrinsic pK(a0) of the N-terminal peptide, good agreement between modeling and experiment is achieved for peptide models with random secondary structures in the entire pH range. Model mobilities of these peptides appear to be relatively insensitive to the assumed secondary structure.     

Itaconic acid carrier ampholytes for isoelectric focusing.

Commercial carrier ampholytes, obtained by coupling polyethylene polyamines to acrylic acid, exhibit a conductivity minimum in the pH range 5.5-6.5 owing to the lack of appropriate pK values of the polyamine in this pH region. By replacing acrylic with itaconic acid, it has been possible to effect substantial improvements in the pH range 5.5-6.5 as itaconic acid has a pK2 value of 5.45. Upon coupling, the pK of the gramma-carboxyl group remains virtually unaltered. With itoconic acid carrier ampholytes it has been possible to improve the conductivity in the pH range 5.5-6.5 by as much as 400% compared with conventional carrier ampholytes. It is suggected that the commercial products should be supplemented with itaconic acid carrier ampholytes in order to obtain a more uniform conductivity and buffering capacity in the pH range 3-10.     

Characterization of synthetic carrier ampholytes for isoelectric focusing.

The synthesis of carrier ampholytes suitable for isoelectric focusing is described. The mixture of hexamethylenetetramine (HMTA), triethylenetetramine (TETA), tetraethylenepentamine (TEPA) and pentaethylenehexamine (PEHA) ampholytes closely resembles commercial Ampholine, and covers the pH range 3-9.5. We have been able to detect focused ampholytes in a gel slab, taking advantage of their different refractive indices, and to assess their relative amounts along the pH gradient. PEHA ampholytes contain up to 20% of chromophoric structures, with two UV peaks at 368 and 315 nm, in a pH-dependent equilibrium, associated with a very weak nitrogen function having a pK of 1.1. This could be the pK6 of the last amino group in PEHA. However, NMR spectra failed to reveal any nitrogen heterocyclic structure formed during the synthesis. This mixture of ampholytes exhibits good conductivity, produces smooth pH gradients and allows sharp protein separations in the pH range 3-9.5. Their synthesis is very easy and their cost is extremely low. Their availability sould make feasible large-scale preparative isoelectric focusing, and attract more interest to continuous-flow techniques, where large amounts of ampholytes are required.     

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.     

Isoelectric buffers, part 3: determination of pKa and pI values of diamino sulfate carrier ampholytes by indirect UV-detection capillary electrophoresis

Ampholytes with close pK(a) values (i.e., good carrier ampholytes (CAs)) are needed as buffers in pH-biased isoelectric trapping (IET) separations. The syntheses of two families of such good CAs were reported recently. Members of the family of diamino sulfate ampholytes (first series) had pI values in the 5.7 < pI < 9.0 range. Members of the family of quaternary ammonium dicarboxylic acid ampholytes (second series) had pI values in the pI < 4.3 range. To further characterize the diamino sulfate ampholytes, their effective mobilities were measured by indirect UV-absorbance detection capillary electrophoresis in a series of background electrolytes (BGEs) with different pH values. The pK(a) and limiting ionic mobility values of the CAs were obtained by fitting these mobility values, as a function of the pH and the ionic strength of the BGEs, to the theoretical mobility expression. These diamino sulfates complete the list of CAs suitable for IET separations.     

Peak identification in capillary isoelectric focusing using the concept of relative peak position as determined by two isoelectric point markers

In CIEF analysis, sample peaks can be identified by their relative peak positions (RPP) that are determined using only two internal pI markers. The two internal pI marker peaks should bracket, as close as possible, the sample peaks. The RPP values of the sample peaks are then calculated using the pI values, peak positions of the two pI markers, and peak position of the sample. Use of this method can effectively compensate for pH gradient distortions that often occur as a result of salts. Also, as shown by the results of this paper, regardless of the linearity of the pH gradient established by the given carrier ampholytes, sample peaks can be identified within an SD of 0.1 pH unit in RPP (<2% RSD) as long as the sample is run using the same carrier ampholytes and maintaining salt concentrations in the range of 0-15 mM.     

Use of quasi-isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.