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

Institute for Molecular Bioscience, The University of Queensland.

School of Land, Crop and Food and Sciences, The University of Queensland.

Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (α_S1, 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 α_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 α_S1-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 α_S1-casein phosphoforms. Deamidation of N⁴⁷ of α_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.