Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures

The field of proteomics requires methods that offer high sensitivity and wide dynamic range. One of the strategies used to improve the dynamic range is sample prefractionation, such as microsolution isoelectric focusing (IEF). We have modified a commercial solution IEF instrument, the Rotofor, to prefractionate protein mixtures by carrier ampholyte-free solution IEF. The focusing chamber of the Rotofor was divided into several compartments by polyacrylamide membranes with imbedded Immobiline mixtures of specific pH values. When an electric field is applied, each protein migrates to the compartment confined by membranes with pH values flanking its isoelectric point. The approach was demonstrated for the focusing of myoglobin into a predicted compartment, as well as the separation of a complex soluble yeast protein mixture into several distinct fractions. The proteins were dissolved in water or 30% isopropanol. The method is applicable to both gel-based and solution-phase protein identification methods, without the need for further sample preparation.     

用液相等电聚焦电泳纯化藻蓝蛋白亚基

以纯藻蓝蛋白(C-phycocyanin, C-PC)为材料, 采用Rotofor系统进行液相等电聚焦(Liquid-phase isoeletric focusing, LP-IEF)电泳纯化C-PC的α, β亚基, 探讨蛋白质亚基纯化的制备电泳(Preparative eletrophoresis)技术. 结果显示, 样品经2次等电聚焦电泳后, C-PC 的α, β亚基分别浓集在pH=4.9和pH=4.1附近, 平板超薄等电聚焦(Slab ultra thin IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PC α, β亚基. 提示LP-IEF是分离纯化等电点差异蛋白质活性亚基的简便有效的方法.     

Identification of synaptic vesicle, pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing

Synaptic pathology is central in the pathogenesis of several psychiatric disorders, for example in Alzheimer's disease (AD) and schizophrenia. Quantification of specific synaptic proteins has proved to be a useful method to estimate synapitc density in the brain. Using this approach, several synaptic proteins have been demonstrated to be altered in both AD and schizophrenia. Until recently, the analysis of synaptic pathology has been limited to postmortem tissue. In living subjects, these synaptic proteins may be studied through analysis of cerebrospinal fluid (CSF). In an earlier study performed by us, one synaptic vesicle specific protein, synaptotagmin, was detected in CSF for the first time using a procedure based on affinity chromatography, reversed-phase chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chemiluminescence immunoblotting. However, other synaptic proteins were not detectable with this procedure. Therefore, we have developed a procedure including precipitation of CSF proteins with trichloroacetic acid, followed by liquid-phase isoelectric focusing using the Rotofor Cell, and finally analysis of Rotofor fractions by Western blotting for identification of synaptic proteins in CSF. Five synaptic proteins, rab3a, synaptotagmin, growth-associated protein (GAP-43), synaptosomal-associated protein (SNAP-25) and neurogranin, have been demonstrated in CSF using this method. The major advantage of liquid-phase isoelectric focusing (IEF) using the Rotofor cell is that it provides synaptic proteins from CSF in sufficient quantities for identification. This method may also be suitable for identification of other types of trace amounts of brain-specific proteins in CSF. These results demonstrate that several synaptic proteins can be identified and measured in CSF to study synaptic function and pathology in degenerative disorders.     

Identification of the apolipoprotein E4 isoform in cerebrospinal fluid with preparative two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

Apolipoprotein E (apoE) was isolated from human cerebrospinal fluid (CSF) from control individuals and patients with Alzheimer's disease (AD). The purification was performed with preparative two-dimensional electrophoresis (2-DE), involving liquid-phase isoelectric focusing (IEF) in the Rotofor cell in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution in the Mini Whole Gel Eluter. ApoE was characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known change of Cys to Arg in position 112 of the apoE4 isoform was identified. This was detected in CSF from AD patients, reflecting the increased frequency of the apoE4 allele in this population. This peptide was not detected in CSF samples from healty control individuals. The use of this rapid electrophoretic separation in proteomic studies of CSF proteins provides single proteins, such as apoE, of high purity in yields sufficient for characterization by MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a useful tool in the investigation of the pathophysiology of brain disorders such as AD.     

Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis

A novel combination of methods, two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), have been used for the analysis of intact brain-specific proteins in cerebrospinal fluid (CSF). 2D-LPE is especially useful for isolating proteins present in low concentrations in complex biological samples. The proteins are separated in the first dimension by liquid-phase isoelectric focusing (IEF) in the Rotofor cell and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the Preparative cell. The removal of SDS by chloroform/methanol/water, followed by sample preparation with the addition of n-octylglucoside, easily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins. Further characterization by proteolytic digestion is also demonstrated. The knowledge of both the molecular weights of the protein and of the proteolytic fragments obtained by peptide mapping increases specificity for protein identification by searching in protein sequence databases. Two brain-specific proteins in human CSF, cystatin C and transthyretin, were isolated in sufficient quantity for determination of the mass of the whole proteins and their tryptic digest by MALDI-TOFMS. This approach simplified the interface between electrophoresis and MALDI-TOFMS.     

Parallel processing in the isoelectric focusing chip

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.     

Evaluation of sample fractionation using micro-scale liquid-phase isoelectric focusing on mass spectrometric identification and quantitation of proteins in a SILAC experiment

Mass spectrometric methods based on stable isotopes have shown great promise for identification and quantitation of complex mixtures. Stable isotope labelling by amino acids in cell culture (SILAC) is a straightforward and accurate procedure for quantitation of proteins from cell lines, that are cultured in media containing the natural amino acid or its isotopically labelled analogue, giving rise to either 'light' or 'heavy' proteins. The two cell populations are pooled and treated as a single sample, which allows the use of various protein purification methods without introducing errors into the quantitative analysis. The quantitation of the proteins is based on the intensities of the light and heavy peptides. The increased number of peptides in a quantitative experiment arising from peptide pairs implies that prefractionation is critical prior to liquid chromatography/mass spectrometric (LC/MS) analysis to minimise signal suppression effects and errors in measurements of the intensity ratios. In this study, the effect of a prefractionation step on identification and quantitation of proteins in a SILAC experiment was evaluated. We show that micro-scale liquid-phase isoelectric focusing in the Micro Rotofor separates proteins into well-defined fractions and reduces the sample complexity. Furthermore, the fractionation enhanced the number of identified proteins and improved their quantitation.     

Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.

     

Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.     

Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins.

Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady-state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate.     

Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9-11 interval

As a last part of an investigation on all 2-pH-unit intervals of carrier ampholytes (CAs) for IEF (see Electrophoresis 2006, 27, 3919-3934; 2006, 27, 4849-4858; 2007, 28, 715-723) two different lots of Servalyt CAs, in the pH 9-11 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by capillary electrophoresis mass-spectrometry of 10 out of 20 fractions harvested, in the second dimension. The findings: the two lots contain 65 and 69 different M(r) compounds, in the M(r) interval of 232-667 Da, for a total of 341-387 isoforms, respectively. Since this is a chaotic organic synthesis, the high reproducibility (here demonstrated for the first time during the 40 years of existence of CAs) of the synthetic process (for two batches produced at 6 years of distance) is remarkable, considering that a 94% agreement for the individual chemicals and 88% agreement for the total number of isoforms for the two lots is found. It is additionally demonstrated that the lower pI species are accompanied by considerably more isoforms than the high pI forms and that in all cases such isoforms consist of family of compounds clustered around the pI of the parental form, with a pI spread of ca. 0.1-0.2 pH units.     

Crystallization of chicken liver (basic) fatty acid binding protein after purification in multicompartment electrolyzers with isoelectric membranes

A preparation of chicken liver (basic) fatty acid binding protein was purified to homogeneity in multicompartment electrolyzers with isoelectric membranes. Large amounts of the isoelectric point (pI) 9.7 protein were collected into a compartment delimited by pI 8.8 and 11.0 membranes. The protein thus purified produced crystals which diffract to higher resolution than those obtained by purification via preparative isoelectric focusing (IEF) in soluble carrier ampholytes. In addition, a novel orthorhombic form with a different molecular packing was obtained. It is hypothesized that, when using conventional IEF, traces of carrier ampholytes could adhere to the protein, particularly in the hydrophobic ligand-binding pocket, rendering the interpretation of the electron density maps difficult. Multicompartment electrolyzers do not present this drawback, since they are based on insoluble buffering species.     

Ion exchange fabric synthesized by graft polymerization and its application to ultra-pure water production

Ion exchange fabric (IEF) having the functional group of sulfonic acid was synthesized by radiation grafting of glycidyl methacrylate on a polyethylene nonwoven fabric and subsequent chemical modification. Total organic carbon eluted from the resulting IEF could be reduced to the concentration less than 1 ppb after washing with organic solvents. Adsorption performance of the obtained IEF was evaluated by 10 ppb Na⁺ solution. The column packed IEF, 7 mm in diameter and 20 mm high, could remove the Na⁺ at the distribution coefficient of 1.2×10⁷ at linear velocity of 400 m/h. At column height of 95 mm, the breakthrough point reached 2.0×10⁵ in bed volume and the degree of column utilization was improved up to 18.7%. From these results, the IEF synthesized by graft polymerization was considered to be applicable for water purification in ultra-pure water production.     

Clinical application of subforms of creatine kinase MM and macro creatine kinases

The subforms of MM isozyme of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) in sera obtained from healthy adults and patients were determined by agarose gel isoelectric focusing (IEF). The patients were classified into six groups according to serum CK-MM activities and IEF patterns. The IEF spectra offered useful information on cell hyperplasia, augmented cell membrane permeability, cell destruction and release time of CK-MM in the circulation from the cells for diagnosis, progress observation and prognosis, especially in the cases of chronic hepatic diseases, acute myocardial infarction and muscular dystrophy. Macro CKs were also determined by IEF. Macro CKs could be completely distinguished from each other, and CK isozymes consisting of macro CK type 1 could be presumed by isoelectric points.     

Characterization of the isoforms of PIXY321, a granulocyte-macrophage-colony stimulating factor-interleukin-3 fusion protein, separated by preparative isoelectric focusing on immobilized pH gradients.

We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence. This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.     

Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples

Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8‐fold.     

Resolving isoforms of aldose reductase by preparative isoelectric focusing in the Rotofor

We have resolved and characterized isoforms of aldose reductase from bovine and porcine lenses by preparative isoelectric focusing with narrow pH gradients using the Rotofor. Both bovine and porcine lens aldose reductases were resolved as two enzyme isoforms. The bovine isoforms were Mr40400 +/- 445 polypeptides of pI4.71 and 5.19. Porcine isoforms were Mr41500 +/- 450 polypeptides of pI 4.90 and 5.30. Staphylococcus aureus V-8 protease digestion patterns for each set of isoforms were essentially identical and all isoforms probably contain blocked amino terminal amino acids. Antiserum to bovine lens aldose reductase cross-reacted with porcine lens aldose reductase. Each isoform displayed substrate preferences characteristic of mammalian aldose reductases. With purification, both bovine and porcine lens aldose reductases became less sensitive to inhibition by 6-fluoro-spiro-(chroman-4.4'-imidazolidine)-2',5'-dione (sorbinil).     

Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post‐separation access to the sample for downstream analysis. The two‐layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post‐separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self‐indexed photomasks for alignment‐free fabrication of multi‐domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.     

Isoelectric focusing in soil science

Applications of isoelectric focusing (IEF) in soil science are illustrated. The materials studied, i. e. humic substances and organic fertilisers, and their behaviour when subjected to IEF are briefly described. Preliminary studies carried out on soil indicate a relation between the isoelectric point and molecular weight, and a connection with humification has been hypothesised; therefore, assessment of soil conditions could be an application of IEF. Another application is evaluation of the stabilisation of organic fertilisers. Finally, IEF can be used to distinguish different organic matrices, as such and in mixtures with mineral fertilisers.     

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.