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 M r of the intact protein. The new protein, with an experimentally determined M r 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 Cys 106 ‐Cys 120 and Cys 66 ‐Cys 161 , as in other mammalian β ‐LGs. The new β ‐LG II variant from donkey was named D. Copyright © 2007 John Wiley & Sons, Ltd.