Fast screening and quantitative evaluation of internally deleted goat α_s1‐casein variants by mass spectrometric detection of the signature peptides

Abstract

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.

The occurrence of genetic polymorphism and related non‐allelic proteins increases the compositional complexity of goat α(s1)‐CN

“…This indicated that the a.a. sequence 59–69 was missing in this sample. Furthermore, the MW value of deleted peptide (f59–69), accounting for 1080 Da, matched exactly the mass difference calculated for B 2 and D 1 variants; the identity of α s1 ‐CN F was confirmed by determination of junction peptides α s1 ‐CN F(f59‐63/43‐63) in the tryptic hydrolysate of the same HPLC peak, consequent to deletion of the sequence (f59–95) from α s1 ‐CN B 2 , as previously found by Picariello et al ; finally, the tryptic peptide map of the third component in the smallest HPLC peak 2 was identical to that of α s1 ‐CN B 2 .…”

Sensitive and Reproducible Intact Mass Analysis of Complex Protein Mixtures with Superficially Porous Capillary Reversed-Phase Liquid Chromatography Mass Spectrometry

“…4,7 These values are above those of other common nonimmunoassay protein detection methods, for example, polyacrylamide gel electrophoresis (PAGE) with silver staining (∼30À 200 fmol), 18 multiple reaction monitoring (MRM) (10À50 amol), 19 or bottom-up protocols such as peptide mass fingerprinting (0.5À5 fmol). 20,21 The sensitivities of the previous online LC/MS top-down platforms were limited by relatively poor RPLC peak resolution, with reported peak widths at half max (w h ) of 17À60 s. These peak widths far exceed the temporal resolution of individual MS scan events (typically 0.1À1.5 s) with detrimental effects on experimental LOD and peak capacity.…”