Heterogeneity of caprine κ-casein macropeptide

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine κ-casein due to the silent substitution Ile/ Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8–14% and 86–92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

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Chromatographic characterization of ovine κ-casein macropeptide

Journal of Dairy Research ◽

10.1017/s0022029900004222 ◽

2000 ◽

Vol 67 (3) ◽

pp. 349-359 ◽

Cited By ~ 23

Author(s):

F. JAVIER MORENO ◽

ISIDRA RECIO ◽

AGUSTÍN OLANO ◽

ROSINA LÓPEZ-FANDIÑO

Keyword(s):

Mass Spectrometry ◽

Acid Phosphatase ◽

Reversed Phase ◽

Free Form ◽

Rp Hplc ◽

Study Heterogeneity ◽

On Line ◽

Molecular Masses ◽

Chromatographic Characterization

Ovine casein macropeptide (CMP) was characterized by anion-exchange FPLC and reversed-phase (RP) HPLC. To study heterogeneity (the degree of glycosylation and phosphorylation), CMP was desialylated with neuraminidase and dephosphorylated with acid phosphatase. Following RP-HPLC, the main CMP components were identified using either on-line or off-line mass spectrometry. The most abundant ovine CMP component was a diphosphorylated carbohydrate-free form, followed by one or two monophosphorylated and a non-phosphorylated asialo-aglyco species. Aglyco non-phosphorylated, monophosphorylated and diphosphorylated forms were in the ratio 3[ratio ]20[ratio ]77. Only ∼ 30% of ovine CMP was glycosylated. Assuming that the monosaccharide fraction of ovine CMP is composed of N-acetylgalactosamine, galactose and N-glycolylneuraminic acid, molecular masses consistent with the presence of CMP containing tetra-, tri-, di- and monosaccharide were identified.

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Determination of Hydrolysis Products of Sulfur Mustards by Reversed-Phase Microcolumn Liquid Chromatography Coupled On-Line with Sulfur Flame Photometric Detection and Electrospray Ionization Mass Spectrometry Using Large-Volume Injections and Peak Compression

Analytical Chemistry ◽

10.1021/ac991035o ◽

2000 ◽

Vol 72 (6) ◽

pp. 1199-1206 ◽

Cited By ~ 34

Author(s):

Edwin W. J. Hooijschuur ◽

Charles E. Kientz ◽

Albert G. Hulst ◽

Udo A. Th. Brinkman

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Large Volume ◽

Reversed Phase ◽

Ionization Mass ◽

Photometric Detection ◽

Hydrolysis Products ◽

Flame Photometric Detection ◽

On Line

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Mass spectrometry-based procedure for the identification of ovine casein heterogeneity

Journal of Dairy Research ◽

10.1017/s0022029900004611 ◽

2001 ◽

Vol 68 (1) ◽

pp. 35-51 ◽

Cited By ~ 23

Author(s):

PASQUALE FERRANTI ◽

ROSA PIZZANO ◽

GIUSEPPINA GARRO ◽

SIMONETTA CAIRA ◽

LINA CHIANESE ◽

...

Keyword(s):

Mass Spectrometry ◽

Analytical Techniques ◽

Reversed Phase ◽

Ionization Mass ◽

Esi Ms ◽

On Line ◽

Protein Components ◽

Rapid Characterization ◽

A Minor ◽

Reference Tools

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization–mass spectrometry (ESI–MS) and matrix-assisted laser desorption ionization–time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HPLC into four major peaks. With ESI–MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography–ESI–MS allowed us to determine each fraction's composition by detecting thirteen αs1-, eleven αs2-, seven β-, and three κ-casein (CN) components. The αs1-CN and αs2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as κ-CN and β-CN. By CE at pH 2·5, each casein fraction was as heterogeneous as that resulting from ESI–MS for the single HPLC-derived fractions. The separation of αs1-CN and αs2-CN proved to be excellent, with the exception of a co-migration of κ0-CN with a minor αs1-CN component and of a glycosylated κ-CN form with low-phosphorylated αs1-CN and β-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI–MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative αs1-CN variants, the non-allelic αs1-CN and αs2-CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

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