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One of the major techniques used for the method development of ternary and quaternary high performance liquid chromatography (HPLC) systems has been to use mixture designs, often referred to as "Glajch's Triangle". This technique does not allow for the systematic and simultaneous optimization of other factors such as gradient time, pH and temperature that affect the quality of separations. An alternative approach is to use experimental designs. The condition, however, that the composition of all components of the mobile phase must total 100% presents a problem when trying to mathematically represent ranges of each mobile phase constituent of a ternary or quaternary system. A method is described here, based on spherical coordinate representations, that adheres to the constraints of the mobile phase composition and allows experimental designs, such as central composite and factorial designs, to be applied to the simultaneous optimization of the mobile phase composition. Other factors, in particular temperature and gradient time, can then be included in the design. As a result of applying these designs to the HPLC separation of phenols and corticosteroids, it was found necessary to include three-way interactions between experimental factors in the model. The significance of these interactions shows that they need to be considered in HPLC method development. 相似文献
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Huang Y Triscari JM Pasa-Tolic L Anderson GA Lipton MS Smith RD Wysocki VH 《Journal of the American Chemical Society》2004,126(10):3034-3035
Analysis of fragmentation patterns from 5654 unique doubly charged tryptic peptides is obtained. Great variability of average relative abundance of bond cleavage is found between different amino acid combinations. There exist similarities as well as differences between b and y ions. Strong enhancement or suppression of cleavage gives insight into possible chemical interactions at reactive conformations formed by preferred phi-psi angles. 相似文献
5.
Anthony Fratiello George A. Vidulich Clifford Cheng Vicki Kubo 《Journal of solution chemistry》1972,1(5):433-444
A competitive solvation study of Al(ClO4)3, Ga(ClO4)3, In(ClO4)3, UO2(ClO4)2, and UO2(NO3)2 in water-acetone-dimethylsulfoxide (DMSO) and water-acetone-hexamethylphosphoramide (HMPT) mixtures has been carried out by direct H1 and P31 nuclear magnetic resonance (NMR) techniques. At low temperature, proton and ligand exchange are slow enough in these systems to permit the observation of signals for bulk and coordinated molecules of water and the organic bases (DMSO and HMPT). Both DMSO and HMPT compete effectively with water for coordination sites in the Al3+, Ga3+, and In3+ systems, with steric effects dominating the HMPT results. Both Al3+ and In3+ are able to bind a maximum of two to three HMPT molecules, for example. In contrast, UO2+ is solvated selectively by the organic molecules to the allowed maximum of 4 molecules per cation. H1 and P31 NMR spectral results support the formation of only the mono-, tri-, and tetra-HMPT solvation complexes. 相似文献
6.
Khaja Basheeruddin Vicki Rothman Simeon Margolis 《Applied biochemistry and biotechnology》1985,11(2):133-140
We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over
the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized
enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg2+. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active
in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by
89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or
membrane-bound enzymes and proteins is discussed. 相似文献
7.
Uncovering the Stoichiometry of Pyrococcus furiosus RNase P,a Multi‐Subunit Catalytic Ribonucleoprotein Complex,by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry 下载免费PDF全文
Xin Ma Lien B. Lai Stella M. Lai Akiko Tanimoto Prof. Mark P. Foster Prof. Vicki H. Wysocki Prof. Venkat Gopalan 《Angewandte Chemie (International ed. in English)》2014,53(43):11483-11487
We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg2+. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg2+‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21?RPP29 and POP5?RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21?RPP29 and (POP5?RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly. 相似文献
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Royston S. Quintyn Mowei Zhou Shai Dagan John Finke Vicki H. Wysocki 《International Journal for Ion Mobility Spectrometry》2013,16(2):133-143
Understanding protein tertiary and quaternary structures is crucial to a better understanding of their biological functions. Here we illustrate for tryptophan synthase that tandem mass spectrometry (MS/MS) reveals not only protein subunit architectures, but also protein unfolding behavior when coupled with ion mobility (IM). In the present study, we verified the subunit arrangement with surface induced dissociation (SID). We are able to correlate experimental results by IM with those obtained in unfolding simulations for the hetero-tetramer Tryptophan Synthase (TS) protein complex by identifying the presence of at least three stable intermediates (I1, I2, and I3) during the unfolding process in collision induced dissociation (CID). We illustrate that the unfolding of the TS complex is likely due to the initial unfolding of an α-monomer subunit, followed by the unfolding of the second α-monomers. We also illustrate the ability of this combination of techniques to not only identify conformational changes of TS upon addition of D,L-α-glycerol phosphate (GP), but also to determine the location of the ligand, which is buried within the α-monomer of the TS. 相似文献
10.
Xin Ma Lien B. Lai Stella M. Lai Akiko Tanimoto Mark P. Foster Vicki H. Wysocki Venkat Gopalan 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2014,126(43):11667-11671
We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg2+. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg2+‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly. 相似文献