title: Proteomic analysis of temperature-dependent changes in stored UHT milk.
abstract: Molecular changes in milk proteins during storage of UHT-treated milk have been investigated using two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF mass spectrometry. UHT-treated samples were stored at three different temperatures, 4 °C, 28 °C, and 40 °C, for two months. Three main changes could be observed on 2-DE gels following storage. They were (1) the appearance of diffuse staining regions above the position of the monomeric caseins caused by nondisulfide cross-linking of α and β-caseins; (2) the appearance of additional acidic forms of proteins, predominantly of α(S1)-casein, caused by deamidation; and (3) the appearance of "stacked spots" caused by lactosylation of whey proteins. The extent of the changes increased with increased storage temperature. Mass spectrometric analysis of in-gel tryptic digests showed that the cross-linked proteins were dominated by α(S1)-casein, but a heterogeneous population of cross-linked forms with α(S2)-casein and β-casein was also observed. Tandem MS analysis was used to confirm deamidation of N(129) in α(S1)-casein. MS analysis of the stacked spots revealed lactosylation of 9/15 lysines in β-lactoglobulin and 8/12 lysines in α-lactalbumin. More extensive analysis will be required to confirm the nature of the cross-links and additional deamidation sites in α(S1)-casein as the highly phosphorylated nature of the caseins makes them challenging prospects for MS analysis.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Australia. j.holland@imb.uq.edu.au
authors.forename: John W
authors.initials: JW
authors.lastname: Holland
authors.forename: Rajesh
authors.initials: R
authors.lastname: Gupta
authors.forename: Hilton C
authors.initials: HC
authors.lastname: Deeth
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000032:analysis / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005519:Food Preservation / Q000379:methods*; D006358:Hot Temperature; D007768:Lactalbumin / Q000032:analysis / Q000737:chemistry; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry; D007785:Lactose / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002364:Caseins; D007782:Lactoglobulins; D008894:Milk Proteins; D007768:Lactalbumin; D007785:Lactose