Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.
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.
UMR 1083 Sciences Pour l'Oenologie, Polyphenol Interaction Research Group, INRA, Bât 28, 2 place Viala, F-34060, Montpellier, France.
Francis
F
Canon
Franck
F
Paté
Emmanuelle
E
Meudec
Thérèse
T
Marlin
Véronique
V
Cheynier
Alexandre
A
Giuliani
Pascale
P
Sarni-Manchado
D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D055232:Proline-Rich Protein Domains; D012471:Salivary Proteins and Peptides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013634:Tannins / Q000737:chemistry*
D012471:Salivary Proteins and Peptides; D013634:Tannins