Peptide mapping by capillary zone electrophoresis: how close is theoretical simulation to experimental determination

A multi-variable computer model is presented for the prediction of the electrophoretic mobilities of peptides at pH 2.5 from known physico-chemical constants of their amino acid residues. The model is empirical and does not claim any theoretical dependencies; however, the results suggest that, at least at this pH, peptides may be theoretically represented as classical polymers of freely joined amino acid residues of unequal sizes. The model assumes that the electrophoretic mobility can be represented by a product of three functions that return the contributions of peptide charge, length and width, respectively to the mobility. The model relies on accurate experimental determination of the electrophoretic mobilities of a diverse set of peptides, by capillary zone electrophoresis (CZE), at 22 degrees C, with a 50 mM phosphate buffer, at pH 2.5. The electrophoretic mobilities of a basis set of 102 peptides that varied in charge from 0.65 to 16 and in size from two to 42 amino acid residues were accurately measured at these fixed experimental conditions using a stable 10% linear polyacrylamide-coated column. Data from this basis set was used to derive the peptide charge, length, and width functions respectively. The main purpose of this endeavor is to use the model for the prediction of peptide mobilities at pH 2.5, and for simulation of CZE peptide maps of protein digests. Excellent agreement was obtained between predicted and experimental electrophoretic mobilities for all categories of peptides, including the highly charged and the hydrophobic. To illustrate the utility of this model in protein studies it was used to simulate theoretical peptide maps of the digests of glucagon and horse cytochrome c. The resulting maps were compared and contrasted with their experimental counterparts. The potential of this approach and its limitations are discussed.     

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.     

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.     

等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定

分泌蛋白质组(secretome)是指在特定的时空条件下,细胞、组织等分泌的全部蛋白质。分泌蛋白质组可能包含了大量的疾病诊断生物标志物,因此其相关研究越来越受到重视。分泌蛋白质组的组成高度复杂且浓度范围宽,这对分析方法提出了挑战。建立有效的蛋白质或肽段预分离策略,将有利于分泌蛋白质的高覆盖率鉴定。本研究以肝癌细胞系MHCC97L的无血清培养分泌蛋白质为研究对象,采用一种新型等电聚焦预分离(OFFGEL)系统,考察了肽段水平的分级对蛋白质鉴定结果的影响。结果表明,分离后各馏分中肽段的等电点分布与理论预测基本一致,每个馏分中单独鉴定的肽段比例接近80%,显示了该系统对肽段的高分辨分离能力。结合生物质谱技术,在肝癌细胞分泌系统中鉴定了2995个蛋白质,显示了该系统在复杂体系蛋白质组研究中的应用潜力。     

Low‐molecular‐weight color pI markers to monitor on‐line the peptide focusing process in OFFGEL fractionation

High‐throughput mass spectrometry‐based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low‐molecular‐weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24‐wells device encompassing the pH range 3–10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI‐TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on‐line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom‐up proteomic approach with or without iTRAQ labeling.     

Isotachophoretic analysis of peptides. Selection of electrolyte systems and determination of purity

Capillary isotachophoresis (ITP) was applied to the qualitative and quantitative analysis of both natural and synthetic oligo- and polypeptides. Based on the mathematical model of acid-base equilibria for a general ampholyte, a procedure and a computer program for the calculation of the pH dependence of the effective and specific charge and effective mobility of peptides with known amino acid sequence were developed which allow the selection of electrolyte systems for peptide isotachophoretic analysis to be rationalized. Basic peptides (bovine pancreatic trypsin inhibitor, bull seminal isoinhibitors of trypsin, arginine vasopressin and adamantylamide-alanylisoglutamine) were analysed with a cationic ITP system at acidic pH. Neutral and acidic peptides (insulin, proinsulin, bull seminal isoinhibitors of trypsin, cow colostrum isoinhibitors of trypsin) were analysed with an anionic ITP system, mostly at alkaline pH. Peptide purity (electrophoretic homogeneity) was determined from the ITP degree of purity defined by a peptide itself and the zone length ratio of its admixtures. Enrichment of peptide in the sample during the purification procedure was measured by its zone length relative to unit mass of the amount of sample analysed.     

pH gradient reversed-phase liquid chromatography as a fractionation tool for the separation of peptides

Recent reports from our laboratory presented a comprehensive theory and demonstrated feasibility of reversed-phase liquid chromatography (RP-LC) employing the programmed gradient of pH of the mobile phase. The aim of that work was to explore the usefulness of the pH gradient-based approach in fractionation of peptides. The experiments were performed on a series of peptides separated at various LC conditions. Retention parameters of peptides in the pH gradient and in the simultaneous pH/organic modifier gradient RP-LC were compared. The best results were obtained with eluents comprising low but constant concentrations of organic modifier while gradient of pH in the mobile phase was developed several times during each chromatographic run. The elaborated LC conditions allowed controlling the elution of peptides not only according to their hydrophobic properties, but also taking into account their electronic properties, represented by isoelectric point (pI) values. The combination of isocratic (regarding organic modifier) LC mode with recurring eluent pH gradient is proposed as an effective fractionation method of peptide mixtures. Moreover, information on hydrophobicity and pI of the peptides, obtained by that approach, might be an additional peptides database matching constraint. Hence, a new tool for analytical and bioinformatics studies of peptides fractionation is proposed.     

Microcapillary liquid chromatography/tandem mass spectrometry using alkaline pH mobile phases and positive ion detection

Extending the dynamic range of microcapillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) peptide sequencing methods is essential for extracting new discoveries from proteomic studies. The complexity of global protein digests and in vivo processed peptide repertoires (as isolated from immunologically important HLA complexes) have led to the development of novel separation methods to increase the number of peptides identified by a single analysis. Separation of complex mixtures by multidimensional high-performance liquid chromatography (HPLC) decreases the number of isolated peptides contained in each fraction and increases the likelihood of detecting low abundant peptides in a background of dominant signals. In this study, we have evaluated the use of two dimensions of reversed-phase chromatography for resolving and sequencing naturally processed HLA-A2 presented peptide repertoires. The first dimension of separation was reversed-phase chromatography using the strong ion pairing reagent trifluoroacetic acid (TFA) to ensure the highest efficiency of peptide fractionation. The second dimension of reversed-phase chromatography was online with an electrospray ionization (ESI) ion trap mass spectrometer. Mobile phases used for the second dimension of chromatography were modified with volatile reagents including a contemporary acetate-modified acidic solvent, which was compared with mobile phases prepared with ammonium hydroxide at an alkaline pH. As expected, we demonstrate improved separation of the HLA-A2 presented fractions using the alkaline pH conditions. However, less obvious was the improved peptide signal-to-noise detected for peptide signals by positive ion ESI ion trap mass spectrometric detection, which was attributed to a reduced chemical background when using the alkaline pH mobile phases that allowed the ion trap to fill with the peptide ions until the automatic gain control detected a full trap. The term 'wrong-way-round ionization' has been used to describe intense [M+H](+) ions generated during ESI under strongly basic solutions. Ultimately, a larger number of the HLA-A2 peptide repertoire was sequenced by coupling TFA-modified reversed-phase fractionation with alkaline-modified microcapillary LC/MS/MS analysis of each fraction. In the present report, we compare the two second-dimension approaches and demonstrate the quality of data that was acquired using alkaline pH reversed-phase conditions.     

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented. The measurements were conducted at 22 degrees C using a 10% linear polyacrylamide-coated column and a 50 mM phosphate buffer at pH 2.5. Excellent separation of peptides and highly reliable peptide maps of protein digests are routinely obtained using these experimental conditions. The electrophoretic data were used to test existing theoretical models that correlate electrophoretic mobility with physical parameters. The results indicate that the Offord model that correlates electrophoretic mobility with the charge-to-size parameter q/M2/3 offers the best fit of our reliable experimental data. Furthermore, we also obtained the capillary zone electrophoretic profile of the endoproteinase Lys-C digests of a peptide sequencing standard, melittin, and horse myoglobin under the same experimental conditions as described above. The resulting peptide maps were compared with corresponding theoretical simulation.     

A Computer Method for Predicting the Electrophoretic Mobility of Peptides

A computer model is presented for the prediction of the electrophoretic mobilities of peptides from physical constants derived from their amino acid sequences. The model assumes that the electrophoretic mobility can be represented by a product of four functions according to the relation:  l_comp= l(L)w(W)q(Q)c(CC), where L (a length parameter) is represented by the number of amino acid residues of the peptide, W (a width parameter) is represented by the average residue mass, Q = the charge of the peptide, and CC = the position of the center of charge relative to the center of mass. The model was used to calculate the electrophoretic mobilities of peptides in a 50 mM phosphate buffer at pH 2.5. Sixty-four test peptides ranging in size from 2 to 39 amino acid residues were used for this study. The calculated mobilities show excellent correlation with experimental measurements with a correlation coefficient greater than 0.98.     

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.     

Evaluation of migration behaviour of therapeutic peptide hormones in capillary electrophoresis using polybrene-coated capillaries

Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and for rapid selection of the optimum pH for separation of mixtures of the modelled compounds. In this work, equations describing the effect of pH on electrophoretic behaviour were used to investigate migration of a series of polyprotic amphoteric peptide hormones between pH 2 and 12 in polybrene-coated capillaries. Polybrene (hexadimethrin bromide) is a polymer composed of quaternary amines that is strongly adsorbed by the fused-silica inner surface, preventing undesired interactions between the peptides and the inner capillary wall. In polybrene-coated capillaries the separation voltage must be reversed, because of the anodic electroosmotic flow promoted by the polycationic polymer attached to the inner capillary wall. The possibility of using polybrene-coated capillaries for determination of accurate ionization constants has been evaluated and the optimum pH for separation of a mixture of the peptide hormones studied has been selected. Advantages and disadvantages of using bare fused-silica and polybrene-coated capillaries for these purposes are discussed.     

Analysis of low-abundance proteins using the proteomic reactor with pH fractionation

We developed a new method consisting of the proteomic reactor coupled with step pH fractionation for the analysis of low-abundance proteins from minute amount of sample. These new reactors were implemented using both SAX and SCX materials. The pH fractions from the SAX reactor provided higher peptide and protein identification than SCX reactor and conventional solution digestion. Interestingly, the physical characteristics (pI, molecular weight, missed cleavage site and grand average hydrophobicity (GRAVY) index, and number of acid and basic amino acid) of the peptides obtained from the SAX and SCX proteomic reactors are drastically different. Furthermore, nearly half of the peptides observed from the pH fractionations from the SAX reactor are of low abundance while only 22% low-abundance proteins are observed with conventional in-solution digestion following 2D LC-MS/MS analysis.     

Use of mixed-mode sorbents for the electrochromatographic separation of thrombin receptor antagonistic peptides

In this study, the thrombin receptor antagonistic peptide TRAP-1 and its alanine-scan analogues, TRAP 2-6, have been employed as probes to characterise the performance of C18/SCX mixed-mode capillary electrochromatographic (CEC) columns. It was found that the resolution of this group of peptides could only be achieved in a narrow pH range with phosphate-based running electrolytes. The influence of the running electrolyte composition, e.g. the buffer choice, the ionic strength, the pH and the organic solvent content, on the electroosmotic flow (EOF) of these mixed-mode CEC columns was investigated. In addition, the retention mechanism for this group of peptide probes in the electrochromatographic process was studied by examining the effect of varying the running electrolyte composition. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving both hydrophobic as well as ion exchange interactions. By modulating the running electrolyte composition, the hydrophobic or ion exchange components of the interaction process could be made to dominate the chromatographic retention of the peptides. Based on this strategy, a high-resolution separation of six closely related synthetic peptides was demonstrated with this mixed-mode CEC system.     

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of peptides and peptide mimetics

Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di- to decapeptides, and phosphinic pseudopeptides--tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH(2)- moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.     

Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.     

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.     

Separation, detection, and identification of peptides by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry at high and low pH

Bioactive peptides and tryptic digests of various proteins were separated under acidic and alkaline conditions by ion-pair-reversed-phase high-performance liquid chromatography (RP-HPIPC) in 200 microm I.D. monolithic, poly(styrene-divinylbenzene)-based capillary columns using gradients of acetonitrile in 0.050% aqueous trifluoroacetic acid, pH 2.1, or 1.0% triethylamine-acetic acid, pH 10.6. Chromatographic performances with mobile phases of low and high-pH were practically equivalent and facilitated the separation of more than 50 tryptic peptides of bovine serum albumin within 15-20 min with peak widths at half height between 4 and 10 s. Neither a significant change in retentivity nor efficiency of the monolithic column was observed during 17-day operation at pH 10.6 and 50 degrees C. Upon separation by RP-HPIPC at high-pH, peptide detectabilities in full-scan negative-ion electrospray ionization mass spectrometry (negESI-MS) were about two to three times lower as compared to RP-HPIPC at low-pH with posESI-MS detection. Tandem mass spectra obtained by fragmentation of deprotonated peptide ions in negative ion mode yielded interpretable sequence information only in a few cases of relatively short peptides. However, in order to obtain sequence information for peptides separated with alkaline mobile phases, tandem mass spectrometry (MS/MS) could be performed in positive ion mode. The chromatographic selectivities were significantly different in separations performed with acidic and alkaline eluents, which facilitated the fractionation of a complex peptide mixture obtained by the tryptic digestion of 10 proteins utilizing off-line, two-dimensional RP-HPIPC at high pH x RP-HPIPC at low pH and subsequent on-line identification by posESI-MS/MS.     

Potential of capillary zone electrophoresis for estimation of humate acid-base properties

Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA(s)) and chernozem (HA(ch)) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA(s) fractions obtained by salting-out with ammonium sulfate at a saturation of 0-40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of "humic hump" was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5-7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA(s) fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA(s) with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the pK(a) of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 x 10(-5) cm(2) V(-1) s(-1) can be used for approximate estimation of HA isoelectric point. The data were discussed and agreement with the random coil structural model has been shown.     

Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling

High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples. Herein, we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography (IPRP×RP 2DLC) strategy for comprehensive proteomic analysis. Both RPLC separation dimensions were performed at low pH, with trifluoroacetic acid(TFA) and formic acid(FA) as mobile phase addictive, respectively. As the good separation resolution offered by ion-pairing effect of TFA, the fractionation efficiency was greatly improved with 74.0% peptides identified in just one fraction. Comparing with conventional high pH RP fractionation, the overall separation rate of IPRP was about 1.6 times that of high-pH RP, which increased the number of identified peptides by 21%. Further, 2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy, exhibiting great potential in clinical proteomics in the future.