The possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect ’signature peptides’, corresponding to the predefined subset of ’proteotypic peptides’, as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the as1-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine as1-CN A and B variants, which differ by a Pro16 (B)->Leu16 (A) substitution. MALDI analysis of the digest allowed the detection of as1-CN f8-22 and caprine as1-CN f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the as1-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.

Toward milk speciation through the monitoring of casein proteotypic peptides

CAIRA, SIMONETTA;FIERRO, OLGA;PINTO, GABRIELLA;G. Picariello;ADDEO, FRANCESCO
2010

Abstract

The possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect ’signature peptides’, corresponding to the predefined subset of ’proteotypic peptides’, as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the as1-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine as1-CN A and B variants, which differ by a Pro16 (B)->Leu16 (A) substitution. MALDI analysis of the digest allowed the detection of as1-CN f8-22 and caprine as1-CN f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the as1-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/366146
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