Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS

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Canon, Francis | Paté, Franck | Meudec, Emmanuelle | Marlin, Therese | Cheynier, Veronique, V. | Giuliani, Alexandre, A. | Manchado-Sarni, Pascale

Edité par CCSD ; Springer Verlag -

Anal. Bioanal. Chem. ISI Document Delivery No.: 522RX Times Cited: 18 Cited Reference Count: 75 Canon, Francis Pate, Franck Meudec, Emmanuelle Marlin, Therese Cheynier, Veronique Giuliani, Alexandre Sarni-Manchado, Pascale French Agence Nationale de la Recherche (A.N.R.) [07-BLAN-0279] The authors thank Helene Boze and Claire Bouchut (UMR 1083 SPO) for their help in protein production and purification and MS experiments. We acknowledge synchrotron SOLEIL and thank all staff for assistance in using beamline DISCO. AG thanks Applied Biosystems (Les Ullis, France) for the loan of the IonCooler Guide. This work is supported by grant 07-BLAN-0279 from the French Agence Nationale de la Recherche (A.N.R.). Springer heidelberg Heidelberg. International audience. Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.

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