A strategy to quantitate global phosphorylation of bone matrix proteins

Anal Biochem. 2016 Apr 15;499:85-89. doi: 10.1016/j.ab.2016.01.017. Epub 2016 Feb 3.

Abstract

Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

Keywords: Extracellular matrix proteins; Global phosphorylation; Human bone; Mouse bone; Osteopontin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aged
  • Aged, 80 and over
  • Animals
  • Bone Matrix / chemistry*
  • Bone Matrix / metabolism
  • Extracellular Matrix / chemistry*
  • Extracellular Matrix / metabolism
  • Extracellular Matrix Proteins / chemistry*
  • Extracellular Matrix Proteins / isolation & purification
  • Extracellular Matrix Proteins / metabolism
  • Female
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Middle Aged
  • Osteopontin / chemistry
  • Osteopontin / deficiency
  • Osteopontin / metabolism
  • Phosphoproteins / analysis*
  • Phosphoproteins / chemistry*
  • Phosphorylation

Substances

  • Extracellular Matrix Proteins
  • Phosphoproteins
  • Spp1 protein, mouse
  • Osteopontin