title: Analysis of bovine milk caseins on organic monolithic columns: an integrated capillary liquid chromatography-high resolution mass spectrometry approach for the study of time-dependent casein degradation.
abstract: Casein proteins constitute approximately 80% of the proteins present in bovine milk and account for many of its nutritional and technological properties. The analysis of the casein fraction in commercially available pasteurized milk and the study of its time-dependent degradation is of considerable interest in the agro-food industry. Here we present new analytical methods for the study of caseins in fresh and expired bovine milk, based on the use of lab-made capillary organic monolithic columns. An integrated capillary high performance liquid chromatography and high-resolution mass spectrometry (Cap-LC-HRMS) approach was developed, exploiting the excellent resolution, permeability and biocompatibility of organic monoliths, which is easily adaptable to the analysis of intact proteins. The resolution obtained on the lab-made Protein-Cap-RP-Lauryl-γ-Monolithic column (270 mm × 0.250 mm length × internal diameter, L × I.D.) in the analysis of commercial standard caseins (αS-CN, β-CN and κ-CN) through Cap-HPLC-UV was compared to the one observe using two packed capillary C4 columns, the ACE C4 (3 μm, 150 mm × 0.300 mm, L × I.D.) and the Jupiter C4 column (5 μm, 150 mm × 0.300 mm, L × I.D.). Thanks to the higher resolution observed, the monolithic capillary column was chosen for the successive degradation studies of casein fractions extracted from bovine milk 1-4 weeks after expiry date. The comparison of the UV chromatographic profiles of skim, semi-skim and whole milk showed a major stability of whole milk towards time-dependent degradation of caseins, which was further sustained by high-resolution analysis on a 50-cm long monolithic column using a 120-min time gradient. Contemporarily, the exact monoisotopic and average molecular masses of intact αS-CN and β-CN protein standards were obtained through high resolution mass spectrometry and used for casein identification in Cap-LC-HRMS analysis. Finally, the proteolytic degradation of β-CN in skim milk and the contemporary formation of low-molecular-weight proteose-peptones (PP) with exact monoisotopic Mr between 9444.0989 Da and 14098.9861 Da was confirmed through the deconvolution of high resolution mass spectra and literature data.
authors.affiliation: Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Roma, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pierri
authors.forename: Dorina
authors.initials: D
authors.lastname: Kotoni
authors.forename: Patrizia
authors.initials: P
authors.lastname: Simone
authors.forename: Claudio
authors.initials: C
authors.lastname: Villani
authors.forename: Giacomo
authors.initials: G
authors.lastname: Pepe
authors.forename: Pietro
authors.initials: P
authors.lastname: Campiglia
authors.forename: Paola
authors.initials: P
authors.lastname: Dugo
authors.forename: Francesco
authors.initials: F
authors.lastname: Gasparrini
mesh_terms: D000818:Animals; D002364:Caseins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008892:Milk / Q000737:chemistry*; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet
chemical_list: D002364:Caseins
keywords: Bovine milk; Capillary liquid chromatography; Caseins; High-resolution mass spectrometry; Organic monoliths