MS-based proteomic characterization of assembled microtubules

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they somehow provide lasting structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. We show that GDP-tubulin, usually considered as an inactive tubulin species, can itself assemble into microtubules preferentially at the minus end, and promote persistent growth. We characterized by mass spectrometry-based proteomics the protein content of tubulin purified from bovine brain (Total) and of microtubules grown from stable GMPCPP seeds in the presence of either GDP-tubulin or GTP-tubulin.