Quantitative proteomic analysis of inorganic phosphate‐induced murine MC3T3‐E1 osteoblast cells

Abstract

Cleavable isotope‐coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate‐treated murine MC3T3‐E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT‐labeled samples were combined, proteolytically digested, and the cICAT‐derivatized peptides isolated using immobilized avidin chromatography. The cICAT‐labeled peptides were resolved into 96 fractions by strong cation‐exchange (SCX) liquid chromatography (LC). Analysis of the SCX‐LC cICAT peptide fractions by microcapillary reversed‐phase LC‐tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate‐responsive gene and was likewise identified as a phosphate‐responsive protein in the current analysis. The 1.76‐fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41‐fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.