Sequence and phosphorylation level determination of two donkey beta-caseins by mass spectrometry

Two coeluting components, with experimentally measured M(r) values of 25529 and 24606 Da, were identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis, the two proteins were identified as donkey beta-CNs and their sequences characterized completely, using the two known beta-CNs from mare as references. The two donkey beta-CNs, showing a mass difference of 923 Da, differ by the presence of the domain E(27)SITHINK(34) in the full-length component M(r) 25529 Da). In comparison with the mare's beta-CNs used as reference, they present nine amino acid substitutions: L -> S(37), R -> H(52), S -> N(81), P -> V(84), L -> V(91), R -> Q(203), P -> L/I(206), L -> F(210) and A -> P(219). Together, these substitutions account for the increase of 18 Da in the M(r) of the donkey beta-CNs with respect to the counterparts from the mare. The molecular mass determination by ESI-MS for the phosphorylated proteins showed that the full-length component was composed of highly multi-phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's beta-CNs, the full-length (226 amino acids) beta-CN was termed variant A, whereas the shorter (218 amino acids) beta-CN was termed variant A(Delta 5).