Isoelectric Focusing pH Gradient Research Articles

Robustness and Ruggedness of Isoelectric Focusing and Superficially Porous Liquid Chromatography with Fourier Transform Mass Spectrometry.

An investigation of a multidimensional proteomics workflow composed of off-gel isoelectric focusing (IEF) and superficially porous liquid chromatography (SPLC) with Fourier transform mass spectrometry (FTMS) was completed in order to assess various figures of merit associated with intact protein measurements. Triplicate analysis performed at both high and low FTMS resolutions on the E. coli proteome resulted in ∼900 redundant proteoforms from 3 to 95 kDa. Normalization of the chromatographic axis to identified proteoforms enabled reproducible physicochemical property measurements between proteome replicates with inter-replicate variances of ±3 ppm mass error for proteoforms <30 kDa, ±1.1 Da for proteins >30 kDa, ±12 s retention time error, and ±0.21 pI units. The results for E. coli and standard proteins revealed a correlation between pI precision and proteoform abundance with species detected in multiple IEF fractions exhibiting pI precisions less than the theoretical resolution of the off-gel system (±0.05 vs ±0.17, respectively). Evaluation of differentially modified proteoforms of standard proteins revealed that high sample loads (100s μgrams) change the IEF pH gradient profile, leading to sample broadening that facilitates resolution of charged post-translational modifications (e.g., phosphorylation, sialylation). Despite the impact of sample load on IEF resolution, results on standard proteins measured directly or after being spiked into E. coli demonstrated that the reproducibility of the workflow permitted recombination of the MS signal across IEF fractions in a manner supporting the evaluation of three label-free quantitation metrics for intact protein studies (proteoforms, proteoform ratios, and protein) over 102-103 sample amount with low femtomole detection limits.

Low-molecular-mass nitrophenol-based compounds suitable for the effective tracking of pH gradient in isoelectric focusing

Low-molecular-mass isoelectric point (pI) markers are a reasonable alternative to commonly used peptide and protein pI markers in the field of isoelectric focusing. We present a comparative study dealing with characterization of 44 nitrophenol-based compounds synthesized by the Mannich reaction which pI values cover a pH range from 3.2 to 10.4. The values of pIs of all the presented nitrophenol-based compounds were precisely determined by capillary isoelectric focusing technique (cIEF) when a standard deviation of measurement reached a value of a few hundredths of a pH unit (n ≥ 3). Differences have been found between in silico analysis of acid-base properties of the investigated compounds and the experimental cIEF data especially at the basic pH region. The data from the acidic pH range showed better correlation between these methods. Only three compounds were excluded from the group of pI markers because they contained impurities originating from the synthesis or long-term storage. Based on the presented results we identified 41 nitrophenol-based pI markers (NPIMs) which can fulfill requirements of the most applications in a field of isoelectric focusing analyses.

Switchable pH actuators and 3D integrated salt bridges as new strategies for reconfigurable microfluidic free-flow electrophoretic separation

We present novel strategies for reconfigurable, high-throughput microfluidic free-flow electrophoretic separation using electrically switchable pH actuators and 3D integrated salt bridges to allow rapid formation of stable pH gradients and efficient electrophoresis. The pH actuator is achieved by microfluidic integration of bipolar membranes which change electrolyte pH by injecting excess H(+) or OH(-) ions produced by a field-enhanced water dissociation phenomenon at the membrane junction upon voltage bias. The technique does not require conventional multiple buffer inflows and leaves no gas production as experienced in electrolysis, thus providing stable pH gradients for isoelectric focusing (IEF) separation. With the pH actuator inactivated, the platform can perform zone electrophoretic (ZE) separation in a medium of constant pH. We also describe the use of 3D integrated ion conductive polymers that serve as salt bridges for improving the voltage efficiency of electrophoresis and to allow high throughput. The proof of concept was successfully demonstrated for free-flow IEF and ZE separation of protein mixtures showing the potential and the simplicity of the platform for high-throughput and high-precision sample separation.

Study on stability mechanism of immobilized pH gradient in isoelectric focusing via the Svensson–Tiselius differential equation and moving reaction boundary

An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson–Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG–IEF and non-IPG–IEF (opposite to IPG–IEF) were developed to unveil stability mechanism of IPG–IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG–IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG–IEF system, (ii) there existed high diffusional current in non-IPG–IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG–IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG–IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG–IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique.

Towards a global analysis of porcine alveolar macrophages proteins through two-dimensional electrophoresis and mass spectrometry

Alveolar macrophages (AM) are the primary phagocytes of the innate immune systems, constituting a link between innate and adaptive immunity. With the aim of studying the porcine AM biology and the dynamics of pig-pathogen cell interactions, we have obtained a reference 2-DE map of the porcine AM proteins. The proteins were separated by 2-DE using a 5–8 range pH gradient in isoelectric focusing and over 800 spots were detected. A set of proteins, covering the pI 5.2–7.4 and M W 19 to 106 kDa ranges, was subjected to MS analysis and 106 proteins were assigned identification by PMF, this identification being confirmed by MS/MS. An important number of proteins is involved in immunological functions, signalling process, transport or apoptosis, confirming that macrophages are involved in a wide range of biological functions. This reference map provides a useful tool for identifying protein pattern changes as a result of inflammation, exposure to infectious agents or genetic diseases.

High Expression of Antioxidant Proteins in Dendritic Cells

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

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.

Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.

Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O2, mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available athttp://www.gartenbau.uni-hannover.de/genetik/AMPP.

A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum.

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

Assessment of the kinetics and sites of reaction of some immobiline chemicals with proteins and peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

A number of proteins and peptides have been incubated with some Immobiline chemicals commonly used in the production of immobilised pH gradients for isoelectric focusing. After various incubation intervals, the resulting reaction mixtures were examined by matrix-assisted laser desorption/ionisation mass spectrometry. At pH 9-10, and after 15-h incubation time, no significant interaction was observed with the two of the investigated proteins which have no Cys residues in their sequences. On the other hand, intense multiple reaction channels were observed with sequences containing a number of Cys residues. The present measurements provide useful information on the kinetics of the reaction and its sensitivity to both the pK(a) of the Immobiline chemicals and the presence of Cys in the investigated sequences. Post source decay measurements on peptides with and without Cys in their sequences provided unambiguous evidence for the involvement of this residue in the reaction conducted at pH 9-10. Possible implications of some of the present deductions for isoelectric focusing separations on immobilised pH gradients are discussed.

Recent developments in preparative free flow isoelectric focusing.

Continuous flow electrophoresis (CFE) is a promising method for preparative fractionation of a variety of biological species, ranging from peptides and proteins to subcellular particles and cells. The high separation efficiency of FFE may be deteriorated by hydrodynamic distortion of zones due to the omnipresent parabolic laminar flow profile. We show in this paper that the detrimental hydrodynamic distortion of separated proteins zones can be reduced, with resultant enhancement of separation efficiency, by employing continuous isoelectric focusing in pH gradients as the actual working regime in an advanced instrumentation. Newly developed media for fast generation of narrow- or broad-range pH gradients under CFE conditions are described. The separation efficiency of these pH gradients is comparable to that of the gradients formed with the aid of synthetic carrier ampholytes. The new media are defined mixtures of nontoxic chemicals, and thus they are compatible with the requirements of human medicine. Experimental data are given showing that the new media offer fractionation of isoforms of proteins, that they offer resolution of proteins differing in isoelectric point (pI) by less than 0.05 pH units, and that these media inhibit proteins precipitation in experiments with human serum proteins.

Comparison of Milk Proteins Using Preparative Isoelectric Focusing Followed by Polyacrylamide Gel Electrophoresis

The major proteins in milks from bovine, caprine, porcine, and murine animals and from humans were compared using a two-dimensional analysis method. In the first dimension, proteins were separated by their isoelectric points using preparative isoelectric focusing in pH gradient of 3 to 10. Twenty fractions from each sample were then analyzed by urea-PAGE and SDS-PAGE. Two-dimensional gels showed characteristic patterns for each milk. Major bovine milk proteins were identified and used as reference for proteins of other mammals. Additionally, some peptides resulting from plasmin hydrolysis were characterized. Caprine milk proteins showed a pattern similar to that of bovine milk except for the absence of αs1-caseins. α-Lactalbumin of bovine and caprine milks resolved as two bands in an immunoblot using bovine α-lactalbumin antibody. Each band corresponded to normal and glycosylated α-lactalbumin. Human, porcine, and murine milk proteins were totally different from those of ruminant milks on the two-dimensional gels. Two-dimensional analysis using preparative isoelectric focusing, followed by PAGE, was a useful method to compare major milk proteins in several mammals because of the rapid simultaneous separation into 20 fractions. This fractionation allows additional analytical procedures for more efficient comparison of chemical and physical properties of the proteins.

PH gradient simulator for electrophoretic techniques in a windows environment

A new program is presented for optimizing mixtures of buffers and titrants for creating pH gradients for isoelectric focusing in immobilized pH gradients and in general for chromatographic processes. The program is written on a windows platform and it includes several novel features compared with previous simulators. First, the estimation of a pH gradient (a non-linear problem) has been transformed into a linear programming problem, thus allowing the use of the simplex as an optimization algorithm. Second, several types of pH gradients can be simulated and optimized, including linear, exponential, logarithmic and sigmoidal. Finally, an equation has been implemented in the program that accounts for the variation of the activity coefficients of ions as a function of the prevailing ionic strength in solution. This simulator was checked experimentally by eluting solutions from a two-vessel gradient mixer and verifying the shape of the various pH gradients. An excellent correlation was found between simulated and experimental data. The program allows the calculation and optimization of pH profiles (including the accompanying ionic strength and buffering power values) for mixtures of up to 50 different monoprotic or oligoprotic buffers and titrants. Calculations and optimizations are performed in a fraction of the time required by previous programs (often in less than 1 min, even for highly complex mixtures).

Immobilized pH gradients (IPG) simulator ‐ an additional step in pH gradient engineering: II. Nonlinear pH gradients

While in the companion paper (Tonani, C. & Righetti, P. G., Electrophoresis 1991, 12, 1011-1021) we gave the general outline of our new computer program, immobilized pH gradients (IPG) simulator, able to simulate and optimize linear pH gradients for isoelectric focusing in immobilized pH gradients, in the present report we extend the application of such a program to: (i) convex exponential gradients, (ii) logarithmic and (iii) polynomial gradients. Such gradients are meant to give equal space to protein spots in complex protein mixtures (e.g., cell lysates, biological fluids) and follow the statistical distribution of protein pI values along the pH axis. They will prove of fundamental importance in two-dimensional maps, both because they optimize the spreading of spots in the two-dimensional plane and because of the excellent reproducibility of immobilized pH gradients. The following concave exponential recipes are given: pH 3-8, pH 3-9, pH 3-10, pH 3-11, pH 4-7, pH 4-8, pH 4-9, pH 4-10, pH 4-11, pH 5-8, pH 5-9, and pH 5-10, as well as the most extended pH 2.5-11 interval. Two interesting logarithmic gradients are described: pH 3-6 and pH 3-7 and one sigmoidal (derived with a polynomial of 5th degree): pH 3-11.

Carrier ampholyte-mediated oxidation of proteins in isoelectric focusing

It has been found that all commercially available carrier ampholytes (the amphoteric buffers used to create and stabilize the pH gradient in isoelectric focusing, IEF) ( e.g., Ampholine, Pharmalyte) are extensively oxidized by persulphate during gel polymerization. These oxidized species are formed for all pH ranges, from acidic to basic, and exhibit chromophoric peaks in the 300–360 nm range. It is believed that these oxidized products are N-oxides (possibly even nitrosamines). Owing to their high redox potentials during IEF, especially in alkaline pH ranges, they are able to capture two H + and two electrons and be reduced, while oxidizing in proteins SH group of Cys to —S—S— (possibly even to cysteic acid). This generates artefactual band heterogeneity, not present in the starting sample, which could never be eliminated as, on rerunning the zone, additional oxidation will take place. In such cases, the presentation of proof of sample homogeneity to agencies such as the FDA, for recombinant DNA products for human consumption, might be impossible. Such a cause of artefactual sample heterogeneity can be virtually eliminated by simply not exposing the carrier ampholytes to persulphate. In the proposed IEF protocol, “empty” gels are polymerized, washed, dried and reswollen in the desired pH interval.

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135–172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40–45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.

Polymerization kinetics of polyacrylamide gels containing immobilized ph gradients for isoelectric focusing

A set of conditions is described for a reproducible incorporation of Immobiline chemicals into a polyacrylamide gel. 0.047% N,N,N′,N′-Tetramethylethylenediamine, 0.033% ammonium persulphate, pH between 7 and 8 and a polymerization time of 1 h at 50°C result in a very high conversion (84–88%) of monomers into the gel matrix with a minimum dispersion of the relative reactivity ratios among the different Immobiline chemicals. Considerably higher incorporations (93–97%) are obtained when polymerization is performed under anaerobic conditions, but this does not alter the relative reactivity ratios among the Immobiline species and considerably adds to the burden of casting an immobilized pH gradient. The different factors leading to optimal polymerization and highly reproducible Immobiline gels are critically evaluated.

Immobilized pH gradients for isoelectric focusing: Interaction between histones and histone‐like proteins with the charged polyacrylamide matrix

AbstractIt has been found that polyacrylamide matrices containing immobilized pH gradients interact strongly with at least two classes of proteins, histones and the histone‐like, ‘high‐mobility group’ (HMG) chromatin proteins, forming insoluble complexes. The nature of these interactions has been demonstrated to be purely ionical: the complexes are split by high ionic strength (0.5 M NaCl) and by altering the pH (full disaggregation being obtained at pH 5.5 and at pH 11.5). On the basis of these observations, soluble Immobiline polymers have been made, by polymerizing them in the absence of cross‐linker (N,N′‐methylenebisacrylamide). Several classes of Immobiline polymers can be produced: a) mixed‐type polymers, containing a blend of basic and acidic Immobilines, in such ratios as to obtain any desired pH in solution; b) homopolymers with a pure carboxyl surface; and c) homopolymers with a pure amino surface. These polymers can be synthesized as ‘mono‐pK species’ or as ‘multiple‐pKcoils’, the latter providing a full range of charges over an ample pH‐interval. It has been found that ‘carboxyl’ Immobiline homopolymers preferentially interact with the HMGl component among the HMG class, thus allowing the partial purification from a lysate by a simple precipitation step.

Comparison of the polypeptide composition of cystic fibrosis plasma with normal plasma by high resolution electrophoresis

Cystic fibrosis and normal plasma proteins were compared by the method of high resolution two-dimensional electrophoresis in polyacrylamide gels. For the first dimension, samples were treated with urea and dithiothreitol and then subjected to isoelectric focusing in pH gradients of 3.5–10.0 or 5.0–8.0. The second dimension involved sodium dodecyl sulfate electrophoresis into gels of 10–20% polyacrylamide gradients. In one “blind” experiment, an attempt to segregate 12 plasma samples into six cystic fibrosis and six normals based on the polypeptide patterns was unsuccessful. Experiments using known cystic fibrotic and normal plasma samples (three further samples of each), depleted of albumin prior to electrophoresis, also failed to confirm the presence of cystic fibrosis related proteins or peptides. We have not been able to substantiate, by electrophoretic means, changes of sialic acid content of any plasma glycoprotein which might be expected to occur, for example, as a result of the postulated altered sialyltransferase activity in cystic fibrosis liver.