Hydrolytically stable, diaminocarboxylic acid-based membranes buffering in the pH range from 6 to 8.5 for isoelectric trapping separations

Diaminocarboxylic acid carrier ampholytes, such as L-histidine, 2,3-diaminopropionic acid, L-ornithine, and L-lysine, were reacted with glycerol-1,3-diglycidyl ether (GDGE) and poly(vinyl alcohol) (PVA) in the presence of sodium hydroxide to produce hydrolytically and mechanically stable hydrogels, supported on a PVA substrate, for use as buffering membranes in isoelectric trapping (IET) separations. The pH values of the DACAPVA membranes were determined with the help of small-molecule pI markers and proteins and were found to be in the 6 < pH < 8.5 range. The membranes were successfully used to isoelectrically trap small ampholytes, desalt ampholyte solutions in IET mode, and effect the binary separation of chicken egg white proteins.     

A family of high-buffering capacity diamino sulfate isoelectric buffers for pH-biased isoelectric trapping separations

pH-biased isoelectric trapping separations are hindered by the lack of suitable isoelectric buffers with pI values in the 5.8 < pI range. Two generic approaches are described here for the cost-effective synthesis of a family of diamino sulfate buffers that have high buffering capacities in their isoelectric state: the first approach relies on the sulfation of existing, commercially available diamino alcohol intermediates, the second approach calls for the synthesis of diamino alcohols from epichlorohydrin and widely available secondary amines, and subsequent sulfation of the new diamino alcohol. The diamino sulfate buffers are recovered in isoelectric state, in high purity. Four members of the family having pI values in the 5.8 < pI < 8.9 range have been synthesized, analytically characterized by capillary electrophoresis (CE), electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), 1-D and 2-D nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. All four diamino sulfates have been successfully used as pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping protein separations.     

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.     

High-buffering capacity, hydrolytically stable, low-pI isoelectric membranes for isoelectric trapping separations

Hydrolytically stable, low-pI isoelectric membranes have been synthesized from low-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker, glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain one amino group and at least two weakly acidic functional groups. The acidic functional groups are selected such that the pI value of the ampholytic component is determined by the pK(a) values of the acidic functional groups. When the concentration of the ampholytic component incorporated into the membrane is higher than a required minimum value, the pI of the membrane becomes independent of variations in the actual incorporation rate of the ampholytic compound. The new, low-pI isoelectric membranes have been successfully used as anodic membranes in isoelectric trapping separations with pH < 1.5 anolytes and replaced the hydrolytically less stable polyacrylamide-based isoelectric membranes. The new low-pI isoelectric membranes have excellent mechanical stability, low electric resistance, good buffering capacity, and long life time, even when used with as much as 50 W power and current densities as high as 33 mA/cm(2) during the isoelectric trapping separations.     

Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixture

The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3相似文献   

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

Free‐flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home‐made carrier ampholyte‐free FFIEF system was constructed via orientated migration of H⁺ and OH^? provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R² = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte‐free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI‐TOF‐MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris‐acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.     

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.     

Extension of separation range in capillary isoelectric focusing for resolving highly basic biomolecules

The non-availability of commercial carrier ampholytes in the pH range greater than 11 has contributed to difficulties in focusing and resolving highly basic proteins/peptides using capillary isoelectric focusing (cIEF). Two different approaches, involving the use of N,N,N',N'-tetramethylethylenediamine (TEMED) and ampholyte 9-11, are investigated for their effects on the extension of separation range in cIEF. The addition of TEMED into pharmalyte 3-10 not only prevents the peptides/proteins from focusing in sections of the capillary beyond the detection point, but also extends the separation range to at least isoelectric point (pI) 12. The combination of ampholyte 9-11 with pharmalyte 3-10 surprisingly provides baseline resolution between bradykinin (pI 12) and cytochrome c (pI 10.3). The sample mixture, containing bradykinin, the high-pI protein calibration kit (pI 5.2-10.3), and cytochrome c digest, is employed to demonstrate the cIEF separation of proteins and peptides over a wide pH range of 3.7-12.     

Sequential injection setup for capillary isoelectric focusing combined with MS detection

Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.     

Fluorescein-based pI markers for capillary isoelectric focusing with laser-induced fluorescence detection

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

Comparison of different capillary isoelectric focusing methods--use of "narrow pH cuts" of carrier ampholytes as original tools to improve resolution

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of "narrow pH cuts" of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.     

Fractionation of carrier ampholytes for isoelectric focusing.

A simple method for fractionating synthetic carrier ampholytes is reported, based on the principle of continuous-flow isoelectric focusing in gel-stabilized layers. An 8% ampholyte solution, encompassing the pH range 3-9.5, is separated into 12 fractions in a chamber filled with Sephadex G-100 by a continuous-flow technique. We are thus able to obtain ampholytes of narrow pH range, encompassing approximately 2 pH units, whose resolving power is comparable with that obtained with commercial Ampholine covering similar pH ranges.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

Diffusion coefficients of proteins in carrier ampholyte versus immobiline gels

The apparent diffusion coefficients of proteins in carrier ampholyte isoelectric focusing (CA-IEF) and in immobilized pH gradients (IPGs) are strongly dependent on the amount of buffering ions present in the system. However, whereas in CA-IEF increased levels of ampholytes facilitate diffusion, in IPGs they strongly quench it. It is concluded that a protein in an IPG matrix is isoelectric but not isoionic, in the sense that it forms a salt with the surrounding ions bound to the polyacrylamide matrix. This salt formation is beneficial as it greatly increases protein solubility at the pI. It is suggested that, when performing zymograms in situ, the IPG gel should contain at least twice the standard amount of Immobiline, so as to keep sharp enzyme bands even with prolonged incubation periods.     

Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients

Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp-41 of the HIV-1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.     

PVA-based tunable buffering membranes for isoelectric trapping separations

PVA-based buffering membranes with tunable pH values were prepared on a PVA substrate by reacting PVA, glycerol-1,3-diglycidyl ether, -NH2 group-containing buffers and -NH2 group-containing titrants in the presence of sodium hydroxide. The pH of the buffering membranes could be tuned in the 3相似文献   

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.     

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3相似文献   

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.