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

• Published in: Journal of proteome research 2008-11, Vol.7 (11), p.5017-5027
• Main Authors: Poth, Aaron G, Deeth, Hilton C, Alewood, Paul F, Holland, John W
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Caseins - analysis; Caseins - chemistry; Caseins - genetics; Electrophoresis, Gel, Two-Dimensional - methods; Humans; Milk, Human - chemistry; Molecular Sequence Data; Phosphorylation; Protein Isoforms - analysis; Protein Isoforms - chemistry; Protein Isoforms - genetics; Proteome - analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr800387s

Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (αS1, β and κ) 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 β-casein was resolved as a train of 6 spots differing in phosphorylation level with 0−5 phosphates attached. To study the less abundant αS1-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 αS1-casein. This included five previously uncharacterized phosphoforms with up to 8 phosphate groups located in two serine-rich tryptic phosphopeptides (27L−R51, 69N−K98) consistent with α-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 β-casein and 9 αS1-casein phosphoforms. Deamidation of N47 of αS1-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.