Native ESI-MS is increasingly used for quantitative analysis of biomolecular interactions. In such analyses, peak intensity ratios measured in mass spectra are treated as abundance ratios of the respective molecules in solution. While signal intensities of similar-size analytes, such as a protein and its complex with a small molecule, can be directly compared, significant distortions of the peak ratio due to unequal signal response of analytes impede the application of this approach for large oligomeric biomolecular complexes. We use a model system based on concatenated maltose binding protein units (MBPn, n = 1, 2, 3) to systematically study the behavior of protein mixtures in ESI-MS. The MBP concatamers differ from each other only by their mass while the chemical composition and other properties remain identical. We used native ESI-MS to analyze model mixtures of MBP oligomers, including equimolar mixtures of two proteins, as well as binary mixtures containing different fractions of the individual components. Pronounced deviation from a linear dependence of the signal intensity with concentration was observed for all binary mixtures investigated. While equimolar mixtures showed linear signal dependence at low concentrations, distinct ion suppression was observed above 20 μM. We systematically studied factors that are most often used in the literature to explain the origin of suppression effects. Implications of this effect for quantifying protein–protein binding affinity by native ESI-MS are discussed in general and demonstrated for an example of an anti-MBP antibody with its ligand, MBP.
The possible inclusion complexes of Cp2NbCl2 into calixarenes hosts have been investigated. The existence of a true inclusion complex in the solid state was confirmed by a combination of NMR, ab-initio calculations, thermogravimetric analysis, FTIR, Raman and PXRD. Ab-initio calculations, 1H NMR solution and solid state 13C CP-MAS NMR results demonstrated that p-sulfonic calix[6]arene does form an inclusion complex with Cp2NbCl2. Raman spectroscopy showed, for the inclusion compound of p-sulfonic calix[6]arene-Cp2NbCl2, a band between 500 and 850 cm−1 characteristic of Nb-O vibration. This result suggests that Nb(V) may engage in coordination with the oxygen of the sulfonate group, as part of the host-guest interaction. However, it is important to mention that the niobocene dichloride (Cp2NbCl2) dissolves in water and undergoes oxidation and hydrolysis processes to yield Cp2NbCl2(OH) species. For that reason this band does not exclude that the Nb-O band belongs to Cp2NbCl2(OH). Solid State 13C CP-MAS NMR and solution 1H NMR spectroscopies together with ab-initio results showed that Cp2NbCl2 is included in the p-sulfonic calix[6]arene cavity, with both Cp rings inside the cavity. In contrast, the solution 1H NMR results demonstrated that calix[6]arene does not form inclusion complex with Cp2NbCl2 in CDCl3 solution. Cp2NbCl2 is not included in the calix[6]arene cavity, possibly due to the lack of sulfonate heads which promote Nb-O interactions and assist the inclusion of Cp2NbCl2 into the cavity. 相似文献
Thin hydrogel films based on an ABA triblock copolymer gelator [where A is pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) and B is biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)] were used as a stimulus-responsive substrate that allows fine adjustment of the mechanical environment experienced by mouse myoblast cells. The hydrogel film elasticity could be reversibly modulated by a factor of 40 via careful pH adjustment without adversely affecting cell viability. Myoblast cells exhibited pronounced stress fiber formation and flattening on increasing the hydrogel elasticity. As a new tool to evaluate the strength of cell adhesion, we combined a picosecond laser with an inverted microscope and utilized the strong shock wave created by the laser pulse to determine the critical pressure required for cell detachment. Furthermore, we demonstrate that an abrupt jump in the hydrogel elasticity can be utilized to monitor how cells adapt their morphology to changes in their mechanical environment. 相似文献
Diastereoselective hydrogenation of 2'-deoxy-2'-exo-methyleneuridine was carried out under homogeneous conditions using a low loading of a chiral Rh catalyst. This, coupled with improvements in the synthesis of the substrate, allowed the smooth pilot plant preparation of the title compound on >10 kg scale. 相似文献