title: Determination of Noncovalent Binding Using a Continuous Stirred Tank Reactor as a Flow Injection Device Coupled to Electrospray Ionization Mass Spectrometry.
abstract: Described is a new method based on the concept of controlled band dispersion, achieved by hyphenating flow injection analysis with ESI-MS for noncovalent binding determinations. A continuous stirred tank reactor (CSTR) was used as a FIA device for exponential dilution of an equimolar host-guest solution over time. The data obtained was treated for the noncovalent binding determination using an equimolar binding model. Dissociation constants between vancomycin and Ac-Lys(Ac)-Ala-Ala-OH peptide stereoisomers were determined using both the positive and negative ionization modes. The results obtained for Ac-L-Lys(Ac)-D-Ala-D-Ala (a model for a Gram-positive bacterial cell wall) binding were in reasonable agreement with literature values made by other mass spectrometry binding determination techniques. Also, the developed method allowed the determination of dissociation constants for vancomycin with Ac-L-Lys(Ac)-D-Ala-L-Ala, Ac-L-Lys(Ac)-L-Ala-D-Ala, and Ac-L-Lys(Ac)-L-Ala-L-Ala. Although some differences in measured binding affinities were noted using different ionization modes, the results of each determination were generally consistent. Differences are likely attributable to the influence of a pseudo-physiological ammonium acetate buffer solution on the formation of positively- and negatively-charged ionic complexes.
authors.affiliation: CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401, Porto, Portugal.
authors.forename: Inês C
authors.initials: IC
authors.lastname: Santos
authors.forename: Veronica B
authors.initials: VB
authors.lastname: Waybright
authors.forename: Hui
authors.initials: H
authors.lastname: Fan
authors.forename: Sabra
authors.initials: S
authors.lastname: Ramirez
authors.forename: Raquel B R
authors.initials: RB
authors.lastname: Mesquita
authors.forename: António O S S
authors.initials: AO
authors.lastname: Rangel
authors.forename: Petr
authors.initials: P
authors.lastname: Fryčák
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Schug
mesh_terms: D000409:Alanine / Q000737:chemistry; D055808:Drug Discovery / Q000379:methods*; D004867:Equipment Design; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods*; D008956:Models, Chemical; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013237:Stereoisomerism; D014640:Vancomycin / Q000737:chemistry
chemical_list: D014640:Vancomycin; D000409:Alanine
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