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

Léonil, Joëlle; Mollé, Daniel; Gaucheron, Frédéric; Arpino, Patrick; Guénot, Pierre; Maubois, J.L.

doi:10.1051/lait:1995314

Cited by 71 publications

(52 citation statements)

References 40 publications

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“…The nature of the linkage of sialic acids in glycoproteins [34] and the sequence Galb(1-.3) GalNAca1-.Ser/ Thr was previously described as a core structure among O-linked saccharides [35]. As regards the glycosylated k-CN forms, the structure determined in this paper is similar to that found in bovine GMP [6,37], except that N-glycolyl-neuraminic acid was present instead of N-acetylneuraminic acid detected in bovine species. In contrast to Moreno et al [33], we did not find the nonphosphorylated GMP.…”

Section: Mass Spectrometric Characterization Of K-cnsupporting

confidence: 65%

“…An obvious method for reducing this complexity is casein fractionation by HPLC. Multiple protein species within the HPLC peak were identified by on-line LC-ESI-MS, though this technique proved inadequately effective for differentiating single phosphorylated components [5,6]. Levels of phosphorylation reflect the activity of the protein kinases and phosphatases which moderate phosphorylation flux in the Golgi apparatus.…”

Section: Introductionmentioning

confidence: 99%

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Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two‐dimensional gel electrophoresis

et al. 2003

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We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis.

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Section: Mass Spectrometric Characterization Of K-cnsupporting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two‐dimensional gel electrophoresis

et al. 2003

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“…The masses of the peptides were determined by HPLC-electrospray ionization (ESI)-MS analysis as follows. Online with the separation by RP-HPLC, a postcolumn flow splitter was used to introduce 10% of the HPLC eluate into the mass spectrometer (25). The ion source voltage was set at 4.8 kV, and the orifice voltage was set at 80 V. The quadruple mass analyzer (Q3) was scanned to an m/z range of 200 to 2,000 Da, with a step size of 0.3 Da and a dwell time of 0.5 ms per step.…”

Section: Methodsmentioning

confidence: 99%

Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

Deutsch

Mollé

Gagnaire

et al. 2000

Appl Environ Microbiol

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The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified ␤-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the ␤-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the ␤-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing ␤-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the ␤-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species. Thermophilic lactobacilli such as Lactobacillus helveticus orLactobacillus delbrueckii along with Streptococcus thermophilus constitute essential lactic starters in Swiss-type cheese (400,000 tons of Emmentaler produced per year worldwide). The three species have a key role in the acidification step and in cheese proteolysis (13). Adequate proteolysis is essential for acceptable quality of the ripened cheese. Whole caseins are first hydrolyzed by milk protease (plasmin) and/or rennet (chymosin and pepsin) into large and intermediate-sized peptides. The proteinases and peptidases from starters subsequently hydrolyze them in small peptides and amino acids, which are known to be aroma precursors. In the three species considered, peptidases are intracellular enzymes released upon lysis in the curd, where they remain active for several weeks (14,40,41). Because of their predominant role in proteolysis, studies on peptidases of thermophilic lactobacilli and S. thermophilus have expanded greatly in recent years (5,18,35,36). Several aminopeptidases, dipeptidases, and peptidases specific to proline-containing peptides (prolidase, prolinase, X-prolyldipeptidyl aminopeptidase, and prolyl aminopeptidase) were isolated, cloned, and sequenced (20); only the general aminopeptidase(s) and the X-prolyl-dipeptidyl-aminopeptidase of thermophilic lactobacilli were shown to be...

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“…A espectrometria de massa tem sido utilizada para determinar a massa molecular das proteínas do leite de vaca 48 , ovelha 49 e cabra 50 , assim como na identificação de variantes genéticas 51,52 , alterações na composição dos aminoácidos e modificações como fosforilação 53 e glicosilação 53 . O estudo das proteínas do leite de vaca utilizando a técnica de HPLC com detecção por espectrometria de massa (MS) forneceu informações importantes para a caracterização das caseínas 52 e outras proteínas do leite 48 .…”

Section: Espectrometria De Massaunclassified

“…O estudo das proteínas do leite de vaca utilizando a técnica de HPLC com detecção por espectrometria de massa (MS) forneceu informações importantes para a caracterização das caseínas 52 e outras proteínas do leite 48 . Trujillo e colaboradores aplicaram igualmente a espectrometria de massa em combinação com HPLC, para a separação em linha, detecção e estudo da composição das proteínas de leite de cabra 49 e de ovelha 50 .…”

Section: Espectrometria De Massaunclassified

Detecção de adulterações em produtos alimentares contendo leite e/ou proteínas lácteas

Veloso¹,

Teixeira²,

Ferreira³

et al. 2002

Quím. Nova

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Recebido em 16/7/01; aceito em 22/10/01 DETECTION OF ADULTERATIONS IN FOOD PRODUCTS CONTAINING MILK AND/OR MILK PROTEINS.A critical review of the most relevant analytical methodologies for quality and authenticity control of dairy products and foods containing milk proteins is presented. Chromatographic, electrophoretic and immunological methods are used for: detection of cow's milk in ewe and goat milks, detection of whey added to milk, detection of caseins and/or whey proteins in non-lactic foods and study compounds resulting from milk proteins degradation. Techniques based on polimerase chain reaction are also suitable for detection of cow's milk on cheeses of ewe and goat milks.Keywords: milk proteins; authenticity; analytical methods. INTRODUÇÃOA autenticidade dos alimentos tornou-se um problema global. É cada vez mais importante detectar a introdução no mercado de produtos fraudulentamente rotulados e de produtos de qualidade inferior, quer por razões econômicas, quer por razões de saúde pública. As adulterações em produtos alimentares contendo leite e/ou proteínas lácteas são relativamente frequentes e diversificadas, incluindo: (i) adição de leite de vaca ao leite de ovelha e/ou cabra para a preparação de queijo; (ii) a incorporação de proteínas do soro na produção de queijos; (iii) a adição de caseínas, ou proteínas do soro a produtos alimentares, particularmente nos derivados de carne.As flutuações sazonais na disponibilidade do leite de cabra e de ovelha e o preço mais elevado comparativamente ao leite de vaca são um incentivo para que os produtores de queijo adulterem os queijos tradicionais, de leite de cabra e de ovelha, com leite de vaca e proteínas do soro [1][2][3][4] . Deste modo, a possibilidade de determinar a matéria prima (tipo de leite) que foi utilizada na produção de queijos, tem grande importância, não só para garantir a genuinidade dos queijos com denominação de origem, e dos queijos fabricados com leites puros, mas também na determinação das percentagens de leite em queijos de mistura. Por outro lado, a incorporação de proteínas do soro em queijos aumenta o rendimento de produção, podendo assim ser utilizadas para substituir a gordura do leite dando origem a queijo de baixo teor de gordura 5 . A adição fraudulenta destas proteínas é normalmente detectada e quantificada pela determinação do glicomacropéptídeo. Este polipeptídeo sendo um componente específico do soro obtido da coagulação do leite pela renina, deverá estar ausente no leite 6,7 . Um outro tipo de fraude que deverá ser detectado é a presença de produtos em desacordo com a rotulagem. Por exemplo, o consumo de produtos que contenham leite e este não seja declarado na rotulagem, poderá causar reações alérgicas, em indivíduos sensíveis a esse produto. As proteínas do soro e as caseínas são frequentemente adicionadas a produtos alimentares devido às suas propriedades agregantes. Assim, por razões éticas e econômicas, torna-se imperativo o desenvolvimento de métodos sensíveis para a detecção dos vários tipos de leites em produto...

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

Cited by 71 publications

References 40 publications

Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two‐dimensional gel electrophoresis

Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two‐dimensional gel electrophoresis

Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

Detecção de adulterações em produtos alimentares contendo leite e/ou proteínas lácteas

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