Fast screening and quantitative evaluation of internally deleted goat as1-casein variants by mass spectrometric detection of the signature peptides.

Currently, the internally deleted caprine as1-casein (as1-CN) variants F and G, associated with lowcasein expression, are detected by means of ordinary descriptive techniques. No relevant procedure isavailable to detect internally deleted goat as1-CN in bulk milks. The availability of full-length andas1-CN F and G variants allowed us to further investigate this issue. Using matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and high-performanceliquid chromatography (HPLC)/electrospray ionization (ESI)-MS and ESI-MS/MS, tryptic signaturepeptides as1-CN F f59-63/f43-63, as1-CN G f4-20/f4-21, as1-CN B2 f4-22 Pro16 and as1-CN A f4-22 Leu16were identified. This also helped to solve the interesting question of how the casein variantscontribute to the composition of goat's bulk milk. Synthetic peptide analogues with ionizationefficiency equivalent to that of tryptic junction peptides were used as internal standards to evaluateas1-CN variants, either individually or globally, using bulk milk from a single goat breed as a modelsystem. Here, as1-CN F accounted for 0.15W0.08% and the as1-CN G variant was missing or below the0.10% detection limit. The analysis of six samples confirmed that as1-CN G was missing and that as1-CN F occurred at a low frequency in hybrid and local breed bulk milks from Mediterranean areas. Inconclusion, a quantitative MS-based application of the signature peptides for full-length andinternally deleted variants in goat's casein is provided. The strategy is also suggested for thedetermination of splice variants in any biological sample.