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Pregnancy-associated glycoproteins (PAGs) are synthesized in the placental cells of ruminants and are detectable in blood, milk, and urine. Many of these proteins have been obtained and characterized from placental extracts by precipitation with 80 % ammonium sulfate. The possibility of purifying PAGs by precipitation with other concentrations of ammonium sulfate remains unexplored. We aimed to study PAG proteins obtained from extracts of ovine placenta at 100 days of gestation through precipitation with 40 % ammonium sulfate (Extract 40). The main protein complex (130 kDa) was obtained after Extract 40 precipitation. Under reducing SDS-PAGE conditions, the 130 kDa complex dissociated in two PAG proteins with apparent molecular weights of 52.1 kDa and 26.1 kDa. The 130 kDa protein appeared to be a molecular complex consisting of two copies of the 52.1 kDa protein linked to one copy of the 26.1 kDa protein, presumably by disulfide bonds. Furthermore, the 52.1 kDa protein consisted of at least three isoforms with distinct isoelectric points. Amino acid microsequencing of the 52.1 kDa protein revealed a chimeric structure containing amino acid sequences of PAG1, PAG4, PAG6, and PAG1-like proteins. This procedure recovered a novel 130 kDa protein complex composed of 26.1 kDa and two 52.1 kDa PAGs. To the best of our knowledge, this has not been previously reported as heterologous polymeric molecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

As a type I Interferon's, Human leukocyte Interferon- alfa (HuIFN-αN3) is a complex pleiotropic molecule composed from several natural subtypes, showing: Antiviral, pro- or anti-inflammatory and antiproliferative activity in vitro. The present work was aimed to detect, analyze and compare the natural subtype's composition of HuIFN-αN3 from EGIS (Budapest, Hungary) or IMZ (Zagreb, Croatia) and to measure theirs pro- or anti-inflammatory, antiproliferative and cytocidal activity in vitro. HuIFN-αN3 from EGIS, (Budapest, Hungary) or IMZ, (Zagreb, Croatia) contains seven "minor" (a-g) and eight "major" (1-8) subtypes with the isoelectric points: 8.12, 7.40, 7.02, 6.90, 6.45, 6.06, 4.45, and 5.80, 5.65, 5.50, 5.30, 5.05, 4.88, 4.72, 4.53. Subtypes show different pro and anti-inflammatory effects. Those separated in the range of pI 8.0 to 6.0 show pro-inflammatory activity. Those separated in the range 5.80 to 3.50 show anti-inflammatory activity. The subtypes isolated from HuIFN- αN3 (IMZ, Zagreb, Croatia) show very weak pro and strong anti-inflammatory activity. The strongest anti- inflammatory activity has subtype 3. The antiproliferative assays on Human Embryonic Fibroblasts (HEF) or Human amnion cells line (FL) cells shows, that the total antiproliferative activity of HuIFN-αN3 is bigger than this from different subtypes, with the exception of subtype a. The cytocidal activity measured as IU/ml needed to get the 50% cytocidal effect on the Adult pig kidney cell line (PLA) cells shows: In the subtype a with 78.1, 1 with 312.5, 2 with 625, 3 with 0, 4 with 1.250, 5 with 0, 6 with 2.500, 7 with 625 and 8 with 0. When different HuIFN-αN3's were tested, this from IMZ (Zagreb, Croatia) and SAV (Bratislava, Slovakia) has 156.2. HuIFN-αN3 from EGIS, (Budapest, Hungary) has 312.5, HuIFN-γ has 156.2, rHuIFN-α1 (recombinant Interferon's) has 2500 and rHuIFN-α2 has 5000. The results show, that all of the subtypes (a-g, 1-8) can be neutralized with the polyclonal anti-IFN-αN3. The values of NI were in the range from:-1.15 till-2.21. It can be concluded that this is the picture of different natural subtypes' content in both preparations from EGIS or IMZ because of different technology. EGIS use concentrated purified preparation, while IMZ use concentrated non purified one. [ABSTRACT FROM AUTHOR]

Isoelectric focusing and chromatofocusing are techniques to separate amphoteric compounds based on their isoelectric points. The commonest used compounds to generate a pH gradient in these techniques are ampholytes. The prohibitively expensive cost of carrier ampholytes limits the application of these techniques in less advantaged parts of the world. A simple and inexpensive method is described in this paper to prepare a broad pH range (pH 3 to 11) ampholytes for use in isoelectric focusing or chromatofocusingin any routine laboratory. The method involves crosslinking polyethylene polyamine to create complex cross-linked polyaminesin the first step. Whereas, the second step involves reacting the cross-linked polyamines with an unsaturated carboxylic acid to produce ampholytes. [ABSTRACT FROM AUTHOR]

Chromatofocusing is investigated as an alternative to protein A chromatography for the initial capture step in a purification process for several monoclonal antibodies and antibody fusion products. For comparison, this work also investigates the use of ion-exchange chromatography with either pH or salt gradient elution as additional alternatives to protein A chromatography. The specific conditions employed for the capture step for the case of chromatofocusing were selected on a rational basis using a computer-aided design method implemented in the form of a Microsoft Excel spreadsheet. Alternative operating conditions were compared experimentally with regard to the product yield achieved as well as the removal of total host cell proteins (HCPs) and of a specific HCP major component. Results from this study indicate that both chromatofocusing and ion-exchange chromatography are useful alternatives to a protein A chromatography capture step in many practical cases. This is especially true for the case of chromatofocusing when it is possible to exploit the ability of the method to create complex gradient shapes that are self-forming inside the column and to simultaneous focus and separate proteins inside the column. [ABSTRACT FROM AUTHOR]

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral 'spiking' studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies. [ABSTRACT FROM AUTHOR]

ABSTRACT Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods. [ABSTRACT FROM AUTHOR]

Simple eluents consisting of tri- or tetrabasic acids solutions (citric and pyromellytic acids and a number of complexon acids-nitriltriacetic (NTA), ethylenediaminetetraacetic (EDTA), cyclohexane-1,2-diamine-N,N,N,N-tetraacetic (CDTA)) or their two-component mixtures were applied for the first time in the chromatofocusing of metal ions on chelating sorbent Tetren-SiO (Silochrom S-120 with bonded tetraethylene pentamine groups). Smoother, quasi-linear pH gradients were formed for eluents; they consisted of citric acid and an EDTA mixture. The separation of Mn, Cr, Co, and Ni were achieved with s simple two-component eluent. [ABSTRACT FROM AUTHOR]

Amino acid-based eluents are used for the chromatofocusing of metal ions on Tetren-SiO chelating sorbent (silica with bonded tetraethylenepentamine groups) for the first time. The smoothest quasilinear pH gradients form for eluents based on glutamic and aspartic acids. The separation of Mn, Cr, Co, Ni, and Cu is achieved. [ABSTRACT FROM AUTHOR]

Highlights: [•] HSA redox isoforms can be separated by external pH gradient chromatofocusing (EPGC). [•] Pharmaceutical HSA and plasma can be analyzed by EPGC. [•] EPGC has advantages over previous analytical and preparative HPLC methods. [Copyright &y& Elsevier]

Highlights: [•] Demonstrated the chromatofocusing of proteins using mixed-mode column packings. [•] Demonstrated the use of additives such as urea and neutral salts for manipulating the retention times of proteins in mixed-mode chromatofocusing. [•] Demonstrated the use of numerical simulation methods for adjusting the pH gradient shape in mixed-mode chromatofocusing. [•] Demonstrated the use of mixed-mode chromatofocusing for blood serum fractionation. [ABSTRACT FROM AUTHOR]

Les auteurs étudient les glycoprotéines dans des moûts de raisins blancs. Ces glycoprotéines sont fractionnées par chromatofocalisation et caractérisées par différentes techniques séparatives : CLHP, électrophorèse, SDS-PAGE, analyse des acides aminés, analyse des oses. Un traitement à la bentonite et un test à la chaleur d'un moût de Sauvignon mettent en évidence qu'une fraction sur les 7 isolées est plus facilement adsorbée par la bentonite et que seulement 3 fractions sur les 7 ont une aptitude à provoquer un trouble après chauffage. La teneur en glycoprotéines varie en fonction du cépage, du degré de maturité de la vendange et des modalités préfermentaires (sulfitage, pressurage et macération).

Although gastropod myoglobin has been extensively studied, there are knowledge gaps in its biological characteristics. We describe for the first time the presence of a myoglobin in the triturative stomach of Biomphalaria gastropods. We compared biochemical parameters of myoglobins of stomach and radular muscles of Biomphalaria glabrata and Biomphalaria tenagophila. Apomyoglobin and holomyoglobin were obtained. Myoglobin was the most abundant protein in the stomach (85.0%) and radular muscles (80.0%) of the two Biomphalaria species evaluated. The Molecular mass and isoeletric point of stomach myoglobins were 16,124.93 Da and 7.98 and 16.095.28 Da and 7.77 for B. glabrata and B. tenagophila, respectively. Stomach myoglobins of B. glabrata and B. tenagophila rate autoxidation were equal to 8.0 × 10−4 h−1 ± 0.0002 and 1.0 × 10−4 h−1 ± 0.00142, respectively. Analysis of N-terminal amino acid sequencing revealed that stomach myoglobins are blocked in this region for a chemical group. Concluding, the differences we observed in the biochemical properties of stomach and radular myoglobins of B. glabrata and B. tenagophila suggest they may be isoform representing an evolutionary event related to the adaptation of these proteins. [ABSTRACT FROM AUTHOR]

Abstract: The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid–base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pK a, and molecular size. By using this approach, variants of representative monoclonal antibodies with isoelectric points of 7.0 or less were separated with high resolution so that the approach can serve as a complementary alternative to isoelectric focusing for characterizing a monoclonal antibody based on differences in the isoelectric points of the variants present. Because the simple buffer system used eliminates the use of polyampholytes, the method is suitable for antibody heterogeneity analysis coupled with mass spectrometry. The method can also be used at the preparative scale to collect highly purified isoelectric variants of an antibody for further study. To illustrate this, a single isoelectric point variant of a monoclonal antibody was collected and used for a stability study under forced deamidation conditions. [Copyright &y& Elsevier]

D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30°C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33°C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38°C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30°C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K and V values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform. [ABSTRACT FROM AUTHOR]

In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized p H gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects. [ABSTRACT FROM AUTHOR]

Citric pulp is an agro-industrial residue from the citrus processing industry with low inorganic phosphorus content applied in animal feed. A new bioprocess was developed to produce and purify a new phytase generated on citric pulp fermentation by Aspergillus niger FS3. The phytase was purified by cationic-exchange, anionic-exchange chromatography and chromatofocusing steps. From SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be 108 kDa. The phytase had an optimum pH of 5.0-5.5 and an optimum temperature of 60°C. The phytase displayed high affinity for phytate, and the K was 0.52 mM. The purified phytase was sufficiently able to withstand pelleting temperatures, retaining sufficiently high phytate-degrading activity. [ABSTRACT FROM AUTHOR]

Abstract: Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way. [ABSTRACT FROM AUTHOR]

Abstract: This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind® D membrane adsorber and HiTrap™ DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind® D membrane at equivalent volumetric throughput and higher capacities than the HiTrap™ DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap™ DEAE FF resin column and was the same for the new AEX membrane and Sartobind® D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput. [Copyright &y& Elsevier]

Abstract: Objective: To investigate whether the glycoform distribution patterns of human chorionic gonadotropin (hCG) obtained by chromatofocusing in pre-evacuation serum are different for patients who will eventually develop into persistent trophoblastic disease in case of complete hydatidiform mole pregnancy as compared to those patients for whom trophoblastic tissue will regress uneventfully. Methods: Pre-evacuation blood samples were collected from women with complete hydatidiform mole with uneventful spontaneous regression after molar evacuation (n =32), from women with complete hydatidiform mole who developed persistent trophoblastic disease after evacuation of their mole (n =28) and, as a control group, from women during the first trimester of normal pregnancy (n =22). The serum specimens were subjected to chromatofocusing, and hCG was determined in the fractions collected in the pH range 7.0–3.0. Results: Receiver operating characteristics (ROC) analysis revealed that 36% of complete hydatidiform mole patients with post-molar persistent trophoblastic disease development had different hCG glycoform profiles at 97% specificity (pH interval 6.3–5.1, hCG cutoff 9.9%). There was a significant difference between complete hydatidiform mole with and without persistent trophoblastic disease for the cumulative percent amounts of hCG in the pH interval 6.3–5.1 (p <0.0003). Conclusion: In 36% of the patients with complete hydatidiform mole with subsequent development of persistent trophoblastic disease, typical glycoform profiles for hCG are observed in pre-evacuation serum samples. This result suggests that hCG glycoform profiles are of potential use in the prediction of persistent trophoblastic disease. [Copyright &y& Elsevier]

Objective: To assess the glycoform distribution patterns of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) during the menstrual cycle at different ages and FSH levels, after menopause, and with premature ovarian failure (POF). Design: Controlled clinical study. Setting: Healthy volunteers in an academic research environment. Patient(s): Women aged 20 to 25 years with normal early follicular (EF) serum FSH (<10 IU/L), women aged 40 to 45 years with normal or increased EF serum FSH, postmenopausal women, and women with POF. Intervention(s): None. Main Outcome Measure(s): FSH and LH glycoform distributions as assessed by chromatofocusing. Result(s): In both postmenopausal and in women with POF, more acidic FSH glycoforms were found compared with young cyclic premenopausal women. In women aged 40 to 45 years with normal FSH levels, these acidic glycoform profiles already showed a statistically significant difference from the younger women. This difference was to attributable to the early follicular and luteal cycle phases. Overall, during aging and after ovarian failure, FSH becomes more acidic, a difference that is already statistically significantly detectable in premenopausal, older women before FSH rises. This is not seen for LH glycoforms. Conclusion(s): The occurrence of postmenopausal-like acidic FSH isoforms precedes the rise of FSH before menopause. [Copyright &y& Elsevier]

Abstract: The technique of “serial displacement chromatofocusing” (SDC) is investigated both theoretically and experimentally with model mixtures of proteins and peptides. The method employs a multistep, retained pH gradient formed using adsorbed buffering species to produce a series of discrete effluent fractions. Each of these fractions may contain several displaced protein bands under conditions of sufficient mass overloading, so that several displacement trains of adjoined bands can be produced in a single chromatographic run. Numerical simulations and experimental results showed selective concentration effects for minor components in a fraction when the feed amount was sufficient large. A computer-aided design method was developed to facilitate the use of the method and was applied to both anion- and cation-exchange column packings. Good agreement was achieved between the designed pH gradients and experimental results. The characteristics of SDC were also explored in terms of its loading capacity, scalability, repeatability, recovery, and differentiation of proteins between their true and apparent isoelectric point values. [Copyright &y& Elsevier]

Abstract: Part I of this study investigated the theory and basic characteristics of “serial displacement chromatofocusing” (SDC). In Part II of this study, SDC is applied to two prototype applications which have potential uses in proteomics and related areas involving the analysis of complex analyte mixtures. In the first application, SDC was used as a prefractionation method prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate a human prostate cancer cell lysate. It was observed that the resolution achieved in narrow-pI-range 2D-PAGE was improved when using SDC prefractionation, so that SDC may be useful as a low-cost, high-speed, and highly scalable alternative to electrophoretic prefractionation methods for 2D-PAGE. The second application involves the use of SDC as the first dimension, and reversed-phase chromatography as the second dimension, to produce a novel, fully automated, two-dimensional high-performance liquid chromatography technique. The method was shown to have performance advantages over one-dimensional reversed-phase chromatography for peptide separations. [Copyright &y& Elsevier]

Abstract: The N-methyl-d-aspartate (NMDA) receptor 2B subunit (NR2B) is important for long-term potentiation (LTP) and synaptic plasticity. The NR2B transgenic mice exhibited larger LTP in the hippocampal CA1 region and enhanced behavioral performance in several learning and memory tasks. In this study, we applied two-dimensional liquid chromatography-based proteomic approach to examine the expression levels of cerebral cortical proteins from 6-month NR2B transgenic (Tg) and their wild-type (WT) mice that were maintained on the same genetic background. Proteins were separated according to the pI in the first dimension using a chromatofocusing column and the hydrophobicity in the second dimension using a nonporous reversed-phase silica column. The DeltaVue software was applied to examine the differential expression of protein samples. Twenty six differentially expressed proteins were identified by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry, including glutamine synthetase (GS), guanine nucleotide-releasing factor 1, carbonic anhydrase, clathrin light chain B (Lcb), enolase 1, ATP synthase, cytochrome c, THO complex 4, and M-phase phosphoprotein 1. The findings were further corroborated in an independent group of NR2B Tg and WT mice by Western blot analysis of two selected proteins. The results revealed a unique profile of cortical proteins in the NR2B transgenic mice. A close association of functional activation of NR2B with the excitatory neurotransmission and neuroplasticity has been discussed. [Copyright &y& Elsevier]

Abstract: The technique of chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: φX174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials. [Copyright &y& Elsevier]

Abstract: Automated two-dimensional liquid chromatography using the PF2D system from Beckman Coulter provides a fractionation platform well suited for differential proteomic studies. To date, the reliability and reproducibility of PF2D has not been accurately tested. Here, we used an optimized software and a pressure-resistant pH electrode, allowing a precise and reproducible control of the pH limits for each fraction during PF2D. We tested the reliability of this improved system by performing several rounds of fractionation using the same protein extract. Three UV maps were generated, leading to 54 chromatograms and more than 3000 protein peaks. Using semi-automated software for peak-to-peak comparison between 2D-LC chromatograms, we demonstrate that the peak concordance is very high. The rates of concordance were higher in the second dimension repeatability tests, indicating that the limiting factors for 2D-LC reproducibility rely on the pI fractionation and sample preparation steps. The reproducibility between maps was closely related to pH curves similarities, further stressing the need of careful pH adjustment and precise electrode calibration. [Copyright &y& Elsevier]

Abstract: pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt. [Copyright &y& Elsevier]

Abstract: The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and α1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2–3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7–8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task. [Copyright &y& Elsevier]

Abstract: The phosphorylation of proteins is a central paradigm of signal transduction. The substitution of neutral hydroxyl groups of serine, threonine and tyrosine with a negatively charged phosphate group alters the physicochemical and immunogenic properties of the protein, which then can be used to isolate these isoforms. In the last decades several different techniques were applied, attempting to selectively enrich protein populations with this post-translational modification. This review aims to give an overview on the arsenal of available methods to extract phosphoproteins focusing on chromatographic approaches. [Copyright &y& Elsevier]

Abstract: Mapping differential expression of soluble proteins has become fairly routine using chromatofocusing in combination with the reversed-phase HPLC (ProteomeLab™ PF-2D by Beckman Coulter Inc.); however, identification of membrane antigens has not been reported thus far. In this report, we demonstrate a targeted proteomic approach employing immunoprecipitation, prior to 2D-LC separation, in tandem with MS/MS that can be used to identify tumor-associated membrane antigens. This system is very sensitive and reproducible in that only 1/4th the amount of starting material is required for analysis as compared to gel-based analysis, and permits a focused environment for eliminating non-specific interactions leading to an accurate resolution of the cognate antigen. This system also circumvents the well-known limitations associated with gel-based approaches. This approach has been validated in the identification of ErB2/HER-2 and was subsequently used to identify CD44E as the cognate antigen for VB1-008, one of our fully human, tumor-specific, monoclonal antibodies. [Copyright &y& Elsevier]

Abstract: The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain. [Copyright &y& Elsevier]

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli . To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I – III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF. iubmb Life , 58: 421–428, 2006 [ABSTRACT FROM AUTHOR]

The formation of decreasing internal pH gradients in carboxylic cation-exchange columns, with a single buffering component in the eluent, was investigated. Two types of carboxylic cation exchangers (hypercrosslinked polystyrene and polymethylmetacrylate based) were used. To develop a pH gradient, the column was pre-equilibrated at pH 7.5 with Tris buffer, followed by washing with different organic acid solutions, including acetic, oxalic, tartaric, citric, and glutamic acids. The smoother quasi-linear gradients in the pH range from 7.5 to 3.5 were obtained with 0.2–1 mM citric acid as eluent. The noticeable role of ionic strength of the starting buffer and of the eluent on the profile of pH gradient was demonstrated. The optimal pH gradient profiles were obtained at equal ionic strengths of both mobile phases. [ABSTRACT FROM AUTHOR]

The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4–4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7–5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil. [ABSTRACT FROM AUTHOR]

A method for analysis of the component composition of multienzyme complexes secreted by the filamentous fungus Trichoderma reesei was developed. The method is based on chromatofocusing followed by further identification of protein fractions according to their substrate specificity and molecular characteristics of the proteins. The method allows identifying practically all known cellulases and hemicellulases of T. reesei: endoglucanase I (EG I), EG II, EG III, cellobiohydrolase I (CBH I), CBH II, xylanase I (XYL I), XYL II, β-xylosidase, α-L-arabinofuranosidase, acetyl xylan esterase, mannanase, α-galactosidase, xyloglucanase, polygalacturonase, and exo-β-1,3-glucosidase. The component composition of several laboratory and commercial T. reesei preparations was studied and the content of the individual enzymes in these preparations was quantified. The influence of fermentation conditions on the component composition of secreted enzyme complexes was revealed. The characteristic features of enzyme preparations obtained in “cellulase” and “xylanase” fermentation conditions are shown. [ABSTRACT FROM AUTHOR]

Abstract: A novel two-dimensional (2D) chromatographic method is developed to separate proteins from malignant breast cancer whole cell lysates. Protein mixtures are first separated according to their pI by chromatofocusing followed by an orthogonal non-porous reversed-phase separation. An important advantage of this 2D chromatographic method is that, unlike gel-based methods, it does not result in methionine oxidation. The lack of methionine oxidation during separation is demonstrated by the analysis of protein tryptic digests using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. Our novel 2D chromatographic method used in combination with on-target light-induced methionine oxidation provides a means for studying methionine-containing peptides. Methionine residues in peptide sequences are partially oxidized with light exposure. Neither the location nor the modification of methionine in the peptide sequence affects the oxidation. As a result, multiple peaks are observed in MALDI-TOF-MS spectra after light exposure. Sequence information derived from light-induced methionine can be applied to enhance the database search results obtained through peptide mass fingerprinting. [Copyright &y& Elsevier]

Recombinant endo-β-1,4-xylanase (Xyl-31rec, 31 kD, pI 8.2-9.3, the tenth family of glycosyl hydrolases) was isolated from the culture liquid of Penicillium canescens (strain with the amplified homologous xylanase gene) by chromatofocusing on Mono P and hydrophobic chromatography on phenyl-Superose. It is shown that the biochemical and kinetic parameters, substrate specificity, stability, and other properties of the recombinant and native enzymes are almost the same. It was found that Xyl-31rec can be used for biobleaching of cellulose, the recombinant P. canescens strains providing a high yield of extracellular Xyl-31rec (up to 800-900 U/ml of culture liquid) and not secreting cellulases. [ABSTRACT FROM AUTHOR]

The composition of the enzyme complex secreted by Penicillium canescens was investigated. A scheme for purification of the main components of the complex by chromatofocusing on a Mono P column was developed. It was found that along with β-galactosidase, the major components of the complex were endo-β-1,4-xylanase (31 kD, pI 8.2-9.3), α-L-arabinofuranosidase (60 kD, pI 7.6), arabinoxylan-arabinofuranohydrolase (70 kD, pI 3.8-4.0), and endo-β-1,3/1,4-glucanase (40 kD, pI 4.4). The substrate specificity, pH and temperature activity optima, adsorbability, thermal stability, and ability for synergic interaction of the isolated enzymes were studied. [ABSTRACT FROM AUTHOR]

A combination of chromatofocusing and gel filtration chromatography resulted in a simple purification of α-amylase from Bacillus licheniformis. The purification was approximately 77-fold. Identification of the purity was established by SDS–PAGE. Molecular weight and isoelectric point of the purified enzyme were 58 kDa and 7.18 respectively. Western blot analysis confirms the specificity of antibody raised against purified α-amylase. [ABSTRACT FROM AUTHOR]

Objective: The aim of the present report was to determine the possible modifications in rat pituitary LH isoforms induced by the spontaneous increase in GnRH at the time of the preovulatory gonadotropin surge. Design: The changes in the quantitative pattern and relative proportions of pituitary LH isoforms in rats on the afternoon of proestrus [INT-P(PM)] were evaluated by comparison with other stages of the estrous cycle (diestrus-1, diestrus-2 and estrus) and ovariectomized (7 and 30 days earlier) animals killed in the morning and in the afternoon of the corresponding day. Methods: The chromatofocusing technique (pH gradient 11.00–7.00) was used to analyze the different molecular species of intrapituitary LH. Results: Pituitary LH from diestrus-1 animals, considered as a baseline pattern in the cycling rat, eluted as 11 isoforms distributed in pH 9.62–8.82, with greater percentages in pH 9.50–9.01. Except for INT-P(PM) pituitaries, there were no major differences in the pattern of LH heterogeneity in the pituitaries of rats from various stages of the cycle. In contrast, significant changes in the charge distribution and relative abundance of LH isoforms were found in the pituitaries from INT-P(PM) rats. INT-P(PM) pituitaries resolved in 16 LH isoforms with a significant shift to less alkaline pIs (pH 9.62–8.11), the more abundant being focused within pH 9.00–8.51. Conversely, a shift to more basic isoforms resulted after ovariectomy, leading to the accumulation of less mature isoforms in the gonadotrope. Conclusions: Presumably, the use of animals on INT-P(PM), at the time of the preovulatory LH surge, made it possible to discriminate such changes in LH isoform distribution. That GnRH, released in association with the rising phase of the LH surge, induces these changes in pituitary LH polymorphism appears to be the most likely possibility. In a previous study we demonstrated that GnRH stimulated galactose incorporation into LH in vitro. In the case of pituitaries from INT-P(PM) rats, the shift toward less alkaline isoforms could potentially result from sialylation of increased terminal galactose.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]