Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations.
Fiche publication
Date publication
février 2017
Journal
Journal of the American Society for Mass Spectrometry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr ENNIFAR Eric
Tous les auteurs :
Wolff P, Da Veiga C, Ennifar E, Bec G, Guichard G, Burnouf D, Dumas P
Lien Pubmed
Résumé
We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate K values (from 0.03 to 0.5 μM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to K(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (K, ΔH) pairs greatly improved the fits and yielded a second K(ITC) close to K(ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of 'low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (K, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments. Graphical Abstract ᅟ.
Mots clés
DNA-polymerase processivity rings, ESI-MS, ITC, Kd determination
Référence
J. Am. Soc. Mass Spectrom.. 2017 Feb;28(2):347-357