Imaging

@article{,

title = {Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations.},

author = {P Wolff and C Da Veiga and E Ennifar and G Bec and G Guichard and D Burnouf and P Dumas},

url = {https://www.ncbi.nlm.nih.gov/pubmed/27957716?dopt=Abstract},

doi = {10.1007/s13361-016-1534-6},

isbn = {27957716},

year  = {2017},

date = {2017-01-01},

journal = {J Am Soc Mass Spectrom},

volume = {28},

number = {2},

pages = {347-357},

abstract = {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 Kd 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 Kd(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 (Kd, ΔH) pairs greatly improved the fits and yielded a second Kd(ITC) close to Kd(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 (Kd, 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.},

keywords = {ARN-MS, DNA-polymerase processivity rings ESI-MS ITC Kd determination, ENNIFAR, Unité ARN},

pubstate = {published},

tppubtype = {article}

}