Phospho-proteomics of DMP-1

Objectives: Dentin matrix protein-1 (DMP-1), an important extracellular matrix protein, acts both as a signaling molecule and a regulator of biomineralization. Phosphorylation plays an essential role in DMP-1 function. Phosphate analyses have shown the phosphorylation level to be at ~60 phosphates/mol. However, the location and number of phosphorylation sites are unknown. Our goal is to utilize DMP-1 either in vitro phosphorylated or purified from mammalian cell lines to analyze the phosphorylation sites by mass spectrometry.

Methods: We purified recombinant DMP-1 (DMP-1) from E. coli and phosphorylated it in vitro using casein kinases I and II (CKI and CKII). The phosphorylation was quantified by 32P radioactivity. In addition, we purified DMP-1 from mammalian U-2 OS (osteoblast) and 293 cells infected with a DMP-1-bearing adenovirus. The phosphorylated DMP-1 was digested with trypsin in-gel and released peptides were analyzed by matrix-assisted time of flight-mass spectrometry (MALDI-TOF-MS). The phosphorylation sites were determined by liquid chromatography- electrospray ionization-ion trap mass spectrometry (LC-ESI-MS/MS).

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Results: The purification from mammalian cells and the in vitro phosphorylation of DMP-1 using CKI and CKII were successful. The results show that CKI is more efficient than CKII in phosphorylating DMP-1. Digestion in vitro of phosphorylated DMP-1 gave the predicted peptide masses by MALDI-TOF-MS analysis. The phosphorylation sites were identified by peak mass shifts of 80 Da and were confirmed by post-source decay analyses. The phosphorylation sites were also detected by LC-ESI-MS/MS. In addition, purification of DMP-1 from both osteoblast and non-osteoblast cell lines was achieved.

Conclusion: We have demonstrated that mass spectrometry can be used to identify the sites of in vitro phosphorylation of DMP-1, as well as its in vivo phosphorylation sites when expressed in mammalian cells. This knowledge will be combined with our signaling and biomineralization studies to better understand the function of DMP-1.