Cow’s milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins (CN) and medium to low abundance whey proteins such as ß-lactoglobulin (ß-LG), a-lactabumin (a-LA), lactoferrin, immunoglobulins, glycoproteins, peptide hormones and enzymes. CNs represent 80% of cow’s milk proteins and possess important nutritional and functional properties, whilst ß-LG (largely unknown function) and a-LA (involved in lactose biosynthesis) represents approximately 10 and 4%, respectively. In addition, a variety of post-translational modifications of these proteins introduces further complexity to the composition of the milk proteome. As genetic variants of CNs and whey proteins influence many properties of milk that are essential to the dairy industry, the development of robust analytical methods for the separation and quantification of variants of milk proteins within single protein fractions is of interest.

Several LC and/or gel (1D- and 2D-PAGE) coupled ESI- and MALDI-based strategies have been applied for the separation and subsequent characterisation of milk protein variants [1-4]. Whilst both top down and bottom up methods could be exploited, the majority of reported analytical workflows have applied bottom-up strategies, due to the high resolution, mass accuracy and the need for large ion fragmentation capability of complex mixtures [1].

This work aims at establishing a robust LC-MS-based method to study intact major proteins from cow’s skim milk. The effects of column temperature, mobile phase composition, and gradient conditions were evaluated to improve protein separation prior to analysis using ESI-Qq-TOF MS. High resolution spectra subsequently allowed accurate mass determination and sequencing of major proteins. This top down LC-MS workflow enables a high-throughput profiling of abundant proteins of cow’s milk with minimal sample preparation.