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Results for “Capillary Isoelectric Focusing”

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7 recordsLinked to original sources

PubMed2024

Deciphering nanoparticle protein coronas by capillary isoelectric focusing-mass spectrometry-based top-down proteomics.

The nanoparticle (NP) protein corona significantly influences the outcome of nanomedicine. We present the first example of top-down proteomics (TDP) measurement of the protein corona using capillary isoelectric focusing-mass spectrometry, identifying seventy proteoforms of 16 cancer-related genes. This technique has the potential to revolutionize our understanding of the protein corona and advance nanomedicine.

Proteomics↗

PubMed2026

Mitigating pH-induced instability in deruxtecan-based ADCs: an onboard-mixing icIEF approach for robust charge heterogeneity characterization.

Accurate charge variant analysis of antibody-drug conjugates (ADCs) is essential for understanding product heterogeneity and ensuring quality control. However, Deruxtecan (DXd)-based ADCs present a unique analytical challenge due to the intrinsic instability of the payload, where the lactone ring readily undergoes hydrolysis under alkaline conditions, resulting in time-dependent shifts in charge distribution during imaged capillary isoelectric focusing (icIEF). In this study, we describe the development of an onboard-mixing icIEF method designed to minimize pH-induced degradation during sample preparation. By separating ADC samples from carrier ampholytes (CAs) prior to injection and enabling real-time mixing within the instrument, this approach effectively suppresses premature lactone ring opening and stabilizes charge variant profiles. Comparative studies between conventional premixing and onboard-mixing approach demonstrated that the latter significantly enhances reproducibility, particularly for acidic variants that are highly sensitive to structural conversion. Comprehensive method validation confirmed excellent precision, linearity, and sensitivity, with consistent performance across run-to-run and intra-day analyses. The results underscore the importance of controlling microenvironmental pH exposure in the analysis of chemically instable ADCs. The proposed onboard-mixing strategy provides a robust and efficient solution for icIEF-based characterization, reducing analytical artifacts while simplifying method development. This approach is broadly applicable to ADCs and other biotherapeutics containing pH-sensitive functional groups.

Hydrogen-Ion Concentration↗

PubMed1976

[Isoelectric focusing of complex protein mixtures in the nanogram range in microgels (author's transl)].

A method is described for isolectric focusing of complex protein mixtures in 2, 5 or 10 mul capillaries. For one separation only 15- 50 ng of a protein mixture is needed. Isoelectric focusing is finished after 10 min, staining takes 20 min and destaining approximately 30 min. Using defined mixtures of Servalyt from different pH ranges, isoelectric focusing can be adapted to the protein sample to be fractionated. Protein peaks separated by isoelectric focusing can be electrophoretically eluted and for further analysis refractionated directly in a microgradient gel. The resolution power of microisoelectric focusing is as good as that of the wellknown macroprocedure, as is demonstrated by isoelectric focusing of the water soluble proteins from cerebellum and heart, of rat and human serum and of a human oncocytoma of the thyroid gland.

Animals↗

PubMed1975

Analytical isotachophoresis in capillary tubes. Analysis of hemoglobin, hemiglobin cyanide and isoelectric fractions of hemiglobin cyanide.

The zone stabilization in capillary isotachophoresis in the water phase has been improved by methylcellulose so that proteins can be analysed. Hemoglobin and hemiglobin cyanide samples were studied as model systems. Ampholine carrier ampholytes were used as spacers, enhancing the detection of the different components. The optimal amounts of Ampholine, however, were found to be much smaller than in most of the previously published reports. Linear relationships were found between the zone lengths and sample amounts, including spacers. The separations were reproducible and reached the isotachophoretic steady state. The hemiglobin cyanide was fractionated by isoelectric focusing. The four main fractions were then analyzed by capillary isotachophoresis and shown to be heterogeneous in mobility with a pH of 7.5 in the leading electrolyte. The component zones of the total hemiglobin cyanide sample were all identified in relation to the isotachophoretic components of the isoelectric fractions. The total analysis time was in average 30-40 min. The sample amounts were about 40 mug protein in each experiment with very small Ampholine volumes, 25-100 nl 40% (w/v).

Cellulose↗

PubMed1979

Macromolecular, anionic pulmonary permeability factor.

Aqueous extracts of fresh or acetone-powdered calf lung have been found to contain a factor which increases the permeability of the microcirculation when injected into the skin of rats. This permeability factor, which is not found in similar extracts of muscle or kidney, was concentrated by Amicon ultrafiltration at a molecular weight range of between 50,000 and 100,000 daltons and via isoelectric focusing at an IEP of pH 4.2. After preparative acrylamide gel electrophoresis, this permeability factor was homogeneous by electrophoretic criteria and in SDS acrylamide gel electrophoresis had a molecular weight of approximately 82,000 daltons. This apparently homogeneous permeability factor from lung was inhibited by pepstatin, and yet possessed no acid proteolytic activity against any substrate. Its activity was completely inhibited by pretreatment of the animals with antihistamines. This pepstatin-inhibitable permeability factor was found largely in the lysosomal fraction of fresh lung and could also be obtained by extraction of large amounts of alveolar "washout" macrophages. Since macrophages, during phagocytosis, are known to leak significant quantities of their lysosomal contents, the fact that one of these components is a material which could release histamine from mast cells and thereby increase locally the permeability of the microcirculation may be of importance in the defense system of the lung.

Animals↗

PubMed1979

Permeability factor contaminating hyaluronidase preparations.

Both crude and highly purified testicular hyaluronidase preparations have been shown to contain a component which increases the permeability of the microcirculation in rat skin. This permeability activity had an isoelectric point of 7.4 while hyaluronidase was 9.4. It also could be separated from hyaluronidase by acrylamide gel electrophoresis. The permeability factor was not inhibited by serum and may explain previous observations that hyaluronidase preparations decrease the ischemia and necrosis appropriate to experimental myocardial infarction in vivo.

Animals↗

PubMed1976

Neutral proteases of human PMN leukocytes with kininogenase activity.

A neutral protease with kininogenase activity was isolated from human polymorphonuclear (PMN) leukocytes by cation exchange chromatography and gel filtration. The protease appears heterogeneous by cation exchange chromatography, isoelectric focusing and cationic disc gel electrophoresis, but homogeneous by gel filtration, sucrose density gradient ultracentrifugation, SDS-disc gel electrophoresis and immunoelectrophoresis. By carrying out the electrophoresis of the protease in acrylamide gels of varying concentrations, it was shown that they represent charge isomers. The protease was stable at pH 4-10, but labile to heat, being almost completely inactivated when incubated for 30 min at 70 degrees C. It exhibited proteolytic activity between pH 5 and 9, being maximal at 7.5-8.5. The molecular weight of the PMN protease was estimated to be about 20,000 daltons by gel filtration in aqueous buffer and about 26,000-28,000 daltons by SDS-disc gel electrophoresis and gel filtration in Sepharose 6B in the presence of the dissociating agent guanidine HCl. Its sedimentation coefficient was about 2.7S. Corresponding to the charge heterogeneity, by isoelectric focusing, the kinin-generating and esterolytic activities of the PMN granule lysate focused between pH 6.0 and 11.5, whereas the isolated PMN protease focused between 10.0 and 11.8. With respect to kinin generation, caseinolysis, and alanine esterase activity, the protease was inhibited by DFP and certain chloromethyl ketone inhibitors, as well as the plasma protease inhibitory a1-antitrypsin, a2-macroglobulin and antithrombin III. Both bradykinin and a methionyl-lysyl-bradykinin-like peptide were generated from highly purified kininogens by a lysosomal lysate containing the PMN protease. However, this assay was done with a crude enzyme preparation which contains an aminopeptidase capable of converting lysyl-bradykinin or methionyl-lysyl-bradykinin to bradykinin. When injected intradermally, the protease induced hyperemia, hemorrhage, and moderate enhancement of vascular permeability, but the mixture of the protease and kininogen induced a marked enhancement of vascular permeability.

Capillary Permeability↗