Use of Mass Spectrometry To Rapidly Characterize the Heterogeneity of Bovine α-Lactalbumin

From a bovine whey protein fraction the nonglycosylated and glycosylated α-lactalbumin fractions were isolated by gel-permeation chromatography followed by reversed-phase high-performance liquid chromatography. Both fractions were studied by electrospray-ionization mass spectrometry (ESI-MS). For the nonglycosylated fraction, a mass of 14 178 Da was found, which was in accordance with the known amino acid sequence of the protein. The glycosylated fraction appeared to be a mixture of components with mass values ranging from ca. 15 840 to 16 690 Da. Given the published carbohydrate composition of the whole glyco-α-lactalbumin fraction, these masses could be matched to those of 14 differently glycosylated forms of α-lactalbumin. Further support for these forms was obtained from the results of a separate mass spectrometric analysis of the mixture of oligosaccharides released from the protein by treatment with peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase F. ESI-MS was found to be a powerful tool to gain insight into the composition of the complex mixture of glycoforms of α-lactalbumin without the need of further purification of these forms or of the oligosaccharides released thereof. Keywords: α-Lactalbumin; (de-)glycosylation; size-exclusion chromatography; reversed-phase HPLC; mass spectrometry