Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry

Phosvitin, derived from the vitellogenin II gene protein, is a highly phosphorylated protein found in egg yolk. A second hypothetical protein has been predicted based on the vitellogenin I gene, but has not been defined at the protein level. Mass spectrometric analysis was used to identify the phosphopeptide sequences and the precise sites of phosphorylation of two phosvitins, phosvitin 1 and phosvitin 2 derived from vitellogenins I and II, respectively. Samples of native phosvitin were subjected to tryptic digestion followed by mass spectrometric analysis: (i) native phosvitin peptides, (ii) after treatment with NaOH, and (iii) after chemical derivatization of P-Ser/P-Thr residues by dithiothreitol under base-catalyzed conditions. A combination of these approaches led to the identification of 68 and 35 phosphopeptides with 89 (81 P-Ser and 8 P-Thr residues) and 62 (57 P-Ser and 5 P-Thr residues) phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. These data for the first time documented on a large scale the major states and sites of phosphorylation of phosvitins with a total of 151 phosphorylation sites. Importantly, the present work also provided the first direct de novo protein amino-acid sequence data for phosvitin 1 protein and evidence for the full expression of vitellogenin I gene.

We have for the first time generated a large number of phosphopeptides (~100) and identified 151 phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. Importantly, this study also led to the discovery of a novel phosvitin 1 and provided the first direct de novo protein amino-acid sequence data for the full expression of vitellogenin I gene. There is considerable interest in naturally occurring phosphopeptides/phosphoproteins and their application in biomedical fields and in the food industry because of their molecular characteristics and non-toxic nature, hence, our work opens new avenues to pursue such endeavors. In addition, the results provide important fundamental biologic information relevant to evolutionary developments of vertebrate animals beginning with the earliest fish, reptiles, birds and more contemporary mammals. For instance, the abundance of phosvitins with a unique degree of phosphorylation in the egg yolk of fish, reptiles, and birds suggests potential biological functions of phosvitins which are critical to the development of embryos of these distant vertebrates.