Detection and sequence determination of a new variant β‐lactoglobulin II from donkey

The sequence determination of a new variant of β-LG II, detected as a minor component by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis of the whey fraction from a milk sample taken from an individual donkey belonging to the ‘Ragusana’ species of eastern Sicily, is reported. Direct RP-HPLC/ESI-MS analysis of the whey fraction from this milk sample allowed the identification of a new variant of β-LG II, based on the determination of the Mr of the intact protein. The new protein, with an experimentally determined Mr of 18311 Da, was detected as a minor component in the whey fraction investigated. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS and RP-HPLC/ESI-MS/MS analyses of the tryptic digest of the new protein demonstrate that it presents two amino acid substitutions with respect to the sequence of β-LG II A, namely a substitution Pro→Cys at position 110, and a substitution Asp→Gly at position 162. The disulfide bonds between the four cysteines, not directly determined in donkey's and horse's β-LG II, were shown to occur between Cys106-Cys120 and Cys66-Cys161, as in other mammalian β-LGs. The new β-LG II variant from donkey was named D. Copyright © 2007 John Wiley & Sons, Ltd.