Detection and sequence determination of a new variant b-lactoglobulin II from donkey Vincenzo Cunsolo, Alessia Costa, Rosaria Saletti, Vera Muccilli and Salvatore Foti * Dipartimento di Scienze Chimiche, Universita ` degli Studi di Catania, Viale A. Doria 6, I-95125 Catania, Italy Received 1 December 2006; Revised 14 February 2007; Accepted 17 February 2007 The sequence determination of a new variant of b-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 b-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 b-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 b-LG II, were shown to occur between Cys 106 -Cys 120 and Cys 66 -Cys 161 , as in other mammalian b-LGs. The new b-LG II variant from donkey was named D. Copyright # 2007 John Wiley & Sons, Ltd. The widespread use of cow’s milk in the human diet has shown that many people are allergic to cow’s milk proteins, which promote immunological adverse reactions. These proteins fall into two groups: caseins, which are a family of phosphoproteins, and whey proteins. Caseins are classified on the basis of their electrophoretic mobility into four groups designed as a-s 1 , a-s 2 , b and k, respectively. 1 All these four types of caseins exhibit an extensive polymorphism due to the effects of post-translational modifications (phosphoryl- ation and glycosylation), alternative splicing of the gene product or genetic polymorphisms. 2 Instead, whey proteins are principally constituted by b-lactoglobulins (b-LG), a-lactoalbumins (a-LA), immunoglobulins and serum albu- min, together with minor components such as lysozyme (LYS) and lactoferrin. The legendary cosmetic and therapeutic properties of equine milk are widely known. 3,4 Particularly in popular tradition, donkey’s milk constitutes a good substitute for cow’s milk in many children with severe protein allergy to cow’s milk. However, these properties still need to be demonstrated objectively at the scientific level. Donkey’s milk is considered to have a composition which is closer to that of human breast milk. Because of its high content of polyunsaturated fatty acids and vitamins, donkey’s milk presents excellent nutritional value and is easily digested. Compared with bovine milk, equine milk presents a very different protein composition. 4,5 The main difference is the proportion of whey proteins in the milk of both species. In fact, in equine milk whey proteins constitute 40–50% of the nitrogen fraction, whereas in bovine milk they represent only 20%. Unfortunately, at the present time equine milk has been less studied than bovine milk and limited data are available for its genetic polymorphism. Major information is available for whey proteins. Sequences of LYS (NCBInr general indexj126613), 6 b-LG I (gij125913) 7 and II (gij125904) 8 from donkey and LYS (gij126614), 9 b-LG I (gij2851437) 10 and II (gij2851599) 11 from horse have been published. More recently, new variants of LYS (named B), b-LG I (named B) and b-LG II (named B and C) from donkey have been identified and characterized. 12 The sequence of three genetic variants of a-LA, named A (gij125991), B (gij125992) and C (gij125992), 13 have also been determined in mare’s milk. In donkey’s milk only one a-LA genetic variant was reported (gij262063), but apparent heterogeneity of the protein was observed. 14 To date, no information is available for the casein fraction in donkey’s milk. Only recently, k-casein has been isolated from mare’s milk and its amino acid sequence has been entirely determined (gij30316368). 15 The primary structures of b- (gij90111821) 16 and a- s1 (gij19031195; gij15723738) 17 caseins in mare’s milk have been deduced from the corresponding cDNA sequences. Recently, advances in proteomics technology have resulted in new powerful tools for the investigation and characterization of complex protein mixtures. In the classical approach, the characterization of protein mixtures is RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2007; 21: 1438–1446 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.2978 *Correspondence to: S. Foti, Dipartimento di Scienze Chimiche, Universita ` degli Studi di Catania, Viale A. Doria, 6, I-95125 Catania, Italy. E-mail: sfoti@dipchi.unict.it Contract/grant sponsor: MIUR (PRIN 2006). Copyright # 2007 John Wiley & Sons, Ltd.