Over the last several years, mass spectrometry, whether used alone or to complement the traditional molecular-based techniques or Edman degradation has become fundamental to the structural analysis of proteins. It is, moreover, virtually irreplaceable in determining post-translational modifications as conventional methods cannot deliver specific or reliable data. When investigating milk proteins, the need for accurate protein sequence analysis has been acknowledged out of the fact that nucleotide sequencing alone is of limited analytical value if not combined with- relevant information regarding the specific protein expressed and the occurrence of phosphorylation, glycosylation and disulphide bridges. What lies at the root of this methodological breakthrough is the development of ionization as a physical process capable of ejecting even the heaviest molecules from polar solutions. Thus, it appears appropriate to state that the new combined mass spectrometric techniques have been established as a valuable and efficient tool for protein and peptide analysis, enabling us therefore to provide accurate information on molecular weight and also to put forth a structural assessment at a low picomole level of material. These techniques offer new possibilities for analysing mixtures of intact proteins, this being of the utmost importance to the study of milk caseins and whey proteins. Suffice it to state that these proteins are known to be part of an extremely complex biochemical system, in which a mixture of components deriving from post- translational processing (phosphorylation, disulphide bridges), the microheterogeneity of glycosylation, and minor components arising from alternative splicing of pre-messenger RNAs may be simultaneously present in extremely small proportions (less than 1%). The technological processes used for dairy products further contribute to the complexity of this system by inducing such phenomena as proteolysis and non-enzymatic glycosylation. Thus, a series of alternative approaches have been developed based on advanced mass spectrometric analysis in conjunction with classic protein chemistry in order to provide an in-depth view of milk protein structure. This review primarily intends to outline several of these novel methodologies as they apply to structural characterization of caseins and whey proteins in fluid milk and in dairy products.
Buy this Article