Analysis of the Human Casein Phosphoproteome by 2-D Electrophoresis and MALDI-TOF/TOF MS Reveals New Phosphoforms

Aaron Poth, Deeth, Hilton C., Alewood, Paul F. and Holland, John W. (2008) Analysis of the Human Casein Phosphoproteome by 2-D Electrophoresis and MALDI-TOF/TOF MS Reveals New Phosphoforms. Journal of Proteome Research, 7 11: 5017-5027. doi:10.1021/pr800387s


Author Aaron Poth
Deeth, Hilton C.
Alewood, Paul F.
Holland, John W.
Title Analysis of the Human Casein Phosphoproteome by 2-D Electrophoresis and MALDI-TOF/TOF MS Reveals New Phosphoforms
Journal name Journal of Proteome Research   Check publisher's open access policy
ISSN 1535-3893
Publication date 2008-11-10
Year available 2008
Sub-type Article (original research)
DOI 10.1021/pr800387s
Open Access Status
Volume 7
Issue 11
Start page 5017
End page 5027
Total pages 11
Editor Hancock, William S
Place of publication United States
Publisher American Chemical Society
Language eng
Subject 090899 Food Sciences not elsewhere classified
C1
860202 Casein
Abstract 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.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
BIOCHEMICAL RESEARCH METHODS
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID UQ11926
Institutional Status UQ

 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 36 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 38 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Sat, 18 Apr 2009, 02:28:04 EST by Emma Cushworth on behalf of Institute for Molecular Bioscience