Fast screening and quantitative evaluation of internally deleted goat alphas1-casein variants by mass spectrometric detection of the signature peptides.

Currently, the internally deleted caprine alphas1-casein (alphas1-CN) variants F and G, associated with low casein expression, are detected by means of ordinary descriptive techniques. No relevant procedure is available to detect internally deleted goat alphas1-CN in bulk milks. The availability of full-length and alphas1-CN F and G variants allowed us to further investigate this issue. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-MS and ESI-MS/MS, tryptic signature peptides alphas1-CN F f59-63/f43-63, alphas1-CN G f4-20/f4-21, alphas1-CN B2 f4-22 Pro16 and alphas1-CN A f4-22 Leu16 were identified. This also helped to solve the interesting question of how the casein variants contribute to the composition of goat's bulk milk. Synthetic peptide analogues with ionization efficiency equivalent to that of tryptic junction peptides were used as internal standards to evaluate alphas1-CN variants, either individually or globally, using bulk milk from a single goat breed as a model system. Here, alphas1-CN F accounted for 0.15+/-0.08% and the alphas1-CN G variant was missing or below the 0.10% detection limit. The analysis of six samples confirmed that alphas1-CN G was missing and that alphas1-CN F occurred at a low frequency in hybrid and local breed bulk milks from Mediterranean areas. In conclusion, a quantitative MS-based application of the signature peptides for full-length and internally deleted variants in goat's casein is provided. The strategy is also suggested for the determination of splice variants in any biological sample.