科研之友
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摘要
Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (alpha(S1), beta and kappa) are a major class of proteins; however, the dynamic range of concentrations in which the various isoforms of each casein exist presents challenges in their characterization. To study human milk casein phosphoforms, we applied traditional two-dimensional polyacrylamide gel electrophoretic (2-DE) separation combined with matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) tandem mass spectroscopic analysis. The abundant beta-casein was resolved as a train of 6 spots differing in phosphorylation level with 0-5 phosphates attached. To study the less abundant alpha(S1)-casein, a cysteine-tagging enrichment treatment was used prior to 2-DE. A train of 9 spots with 4.4 < pI < 5.3 were identified as alpha(S1)-casein. This included five previously uncharacterized phosphoforms with up to 8 phosphate groups located in two serine-rich tryptic phosphopeptides (L-27-R-51, N-69-K-98) consistent with alpha-caseins from various ruminant species. MS/MS analysis of the phosphopeptides released by tryptic digestion enabled identification of the residue-specific order of phosphorylation among the 6 beta-casein and 9 alpha(S1)-casein phosphoforms. Deamidation of N-47 of alpha(S1)-casein was also a feature of the MS analysis. This study represents the first comprehensive analysis of the human casein phosphoproteome and reveals a much higher level of phosphorylation than previously recognized. It also highlights the advantages of 2-DE for examining the global pattern of protein phosphoforms and the limitations of attempting to estimate phosphorylation site occupancies from "bottom-up" studies.
- 出版日期2008-11
