Characterisation of the casein variants in goat bulk milks using on-line RP-HPLC/ESI-MS

Abstract

International audienceA HPLC/ESI-MS method was used to separate and analyse casein variants in goat bulk milks. From mass determination, about 25 casein components were characterised in each milk casein sample with up to 7 compounds obtained in the same HPLC fraction. The different caseins were each detected at various phosphorylation levels. Only casein compounds present at levels higher than 1% of total casein were obtained, as confirmed by the analysis of individual goat milks of known composition. The discrimination threshold obtained for the differentiation of molecular masses was quantified by the variation coefficient (CV = 0.02% ). Assignment of masses to known compounds was made by reference to theoretical masses. Some compounds, in $\kappa$CN, $\alpha_{{\rm s1}}$CN and $\beta$CN fractions could not be assigned. It is possible that they were non-described variants. A limitation of the method was revealed when variants present in the same RP-HPLC fraction had the same molecular mass (as was the case for $\alpha_{{\rm s1}}$CNA and $\alpha_{{\rm s1}}$CNB$_1$) or when their masses differed by ~80 g$\cdot$mol$^{-1}$ (as was the case for $\alpha_{{\rm s2}}$CNA and $\alpha_{{\rm s2}}$CNB). In this latter case, the 2 variants could not be differentiated from the different phosphorylation levels of the same, indeed the addition of a phosphoseryl residue increased the molecular mass by 80 g$\cdot$mol$^{-1}$. To overcome these limitations, an improvement of the method was proposed. In the 3 bulk milks analysed the same main variants, $\kappa$CN-Ile 2P, $\alpha_{{\rm s2}}$CN A11P, $\alpha_{{\rm s1}}$CN A and/or E were found, and the same non-identified component in $\beta$CN fraction. However, differences between the 3 bulk milks analysed were observed. A variation in the relative proportions of variants in $\kappa$CN and $\beta$CN fractions was observed in the different milks and it is proposed, if confirmed, that it could be related to the selection stage of the producing goats. Thus, the HPLC/ESI-MS method applied to bulk milks collected throughout a country allowed for an accurate determination of the casein variants with a higher frequency. The analysis of individual milks would allow a fast and accurate characterisation of the casein phenotype of goats.Caractérisation des variants de la caséine contenue dans des laits de chèvre de grand mélange par RP-HPLC/ESI-MS. Une méthode RP-HPLC/ESI-MS a été utilisée pour la mise en évidence et la caractérisation des différents variants de la caséine présents dans des laits de chèvre de très grand mélange. L'identification des composés à partir des masses expérimentales était réalisée par référence aux masses théoriques des composés connus. Environ 25 composants ont été mis en évidence dans chaque lait. Dans une même fraction, jusqu'à 7 composés ont pu être séparés par leur différence de masse. Les différents niveaux de phosphorylation des caséines étaient individualisés. Les composants de la caséine présents à des teneurs supérieures à 1 % de la caséine totale ont été mi...

Fast screening and quantitative evaluation of internally deleted goat αs1‐casein variants by mass spectrometric detection of the signature peptides

“…The order of elution of the caseins, with crescent t R , was k-< a s2 -< a s1 -< b-casein. In terms of the elution, the overall HPLC pattern of the dephosphorylated casein was similar to that of native casein, 15,18,19 which was confirmed by the fact that the molecular mass values obtained by MALDI-TOF MS were close to those expected from the known amino acid sequences, as reported in Fig. 2.…”

Protein composition and polymorphism in the milk of Skopelos goats

“…In 2 samples out of 25, there was a mass attributed to κ-cn G found in Italian and Turkish breeds [39,50]. The major form of κ-Cn was the 2P form, although the 1P form was also evident, as previously reported [35,38,48].…”

“…The α s2 -cn is the most phosphorylated casein and in most cases it has been found with 7-11P, the most abundant type being 11P, as reported by Pierre et al [38] and Neveu et al [35]. In 4 samples out of 25 analyzed by RP-HPLC/ESI-MS, types with 12 P and 13 P were observed, as reported by Trujillo et al [48].…”