Characterization of phospho‐proteins in bovine and buffalo caseins using atmospheric pressure ionization mass spectrometry

Casetta, Bruno; Bonner, Ron; Shushan, Bori; Fior, G.O.

doi:10.1002/oms.1210270310

Cited by 7 publications

(6 citation statements)

References 1 publication

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“…Thus, it is possible to characterize: i) milk from single cows; ii) pooled milk; iii) a fraudulent alteration of human, goat and sheep milks with bovine milk as already shown by Casetta et al (1992); and iv) proteolysis of caseins or whey proteins. …”

Section: Resultsmentioning

confidence: 99%

Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry

Léonil

Mollé²,

Gaucheron³

et al. 1995

Lait

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Summary -Proteins from skim milk, paracaseinate and whey have been successfully analyzed by reverse-phase high-pertormance liquid chromatography (RP-HPLC) cou pied with electrospray ionization mass spectrometry (ESI-MS). The major milk proteins were identified by comparison of molecular masses determined by ESI-MS to molecular masses calculated from amino acid composition deduced from primary structures and cDNA sequences for some proteins. This method has permilted the simultaneous identification of caseins and whey protein variants. Observed molecular masses of major milk proteins were found to be 19038.0 ± 2.2 Da forK-CN A-l P (number of experiments n = 6); 19007.0 ± 1.1 Da for K-CN B-1P (n = 4); 25230.0 ± 2.1 Da for as2-CN A (n = 9); 23617.2 ± 1.3 Da for as1-CN B-8P (n = 14); 24 092.0 ± 1.7 Da for~-CN B-5P (n = 6); 24 025.3 ± 1.0 Da for~-CN A 1-5P (n = 14); 23 984.8 ± 0.7 Da for~-CN A2_5P (n = 14); 18278.3 ± 2.2 Da for the monomeric form of~-LG B (n = 5); 18 364.8 ± 1.6 Da for the monomeric form of~-LG A (n = 5); 14 179.21 ± 3.14 Da for a-LA (n = 5). Hence an accuracy of 0.01 % was obtained by ESI-MS analysis. It was also shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for studying the proteolysis and determining the specificity of used enzymes in some technological treatments as shown for milk-clotting enzymes.

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Section: Resultsmentioning

confidence: 99%

Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry

Léonil

Mollé²,

Gaucheron³

et al. 1995

Lait

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“…This allows for the exact calculation of molecular mass of the protein which substantially serves as the more usual and the more convenient reference. The molecular mass of a protein can now be obtained rapidly and with high accuracy using mass spectrometry [6,10]. It can be used to characterise compounds with direct reference to their known theoretical masses.…”

Section: Introductionmentioning

confidence: 99%

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

Aimar

Mollé

Zahoute

2001

Lait

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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...

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“…More recently, immunoblotting3 procedures and atmospheric pressure ionization mass spectrometry (API/MS) have been used and, in the latter case, speciÐc bovine and bu †alo caseins have been easily detected. 4,5 In previous investigations, the capabilities of matrixassisted laser desorption/ionization mass spectrometry (MALDI/MS) in the dairy industry were demonstrated. 6 The method allows valid Ðngerprinting of the protein proÐle of cow milk and was employed to test the possible thermal degradation of milk proteins during pasteurization and/or sterilization treatments.…”

Section: Introductionmentioning

confidence: 99%

The potential of matrix-assisted laser desorption/ionization mass spectrometry in the quality control of water buffalo mozzarella cheese

Angeletti

Gioacchini

Seraglia³

et al. 1998

J. Mass Spectrom.

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Adulteration by addition of bovine milk to water buffalo milk employed for mozzarella cheese production is often observed. Water buffalo milk and mozzarella cheese were analysed by matrix‐assisted laser desorption/ionization mass spectrometry in order to achieve their rapid and accurate characterization and to evaluate possible fraudulence in mozzarella cheese production. © 1998 John Wiley & Sons, Ltd.

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Characterization of phospho‐proteins in bovine and buffalo caseins using atmospheric pressure ionization mass spectrometry

Cited by 7 publications

References 1 publication

Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry

Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry

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

The potential of matrix-assisted laser desorption/ionization mass spectrometry in the quality control of water buffalo mozzarella cheese

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