Identification of phosphorylation sites on neurofilament proteins by nanoelectrospray mass spectrometry

Neurofilament (NF) proteins are intermediate filaments found in the neuronal cytoskeleton. Phosphorylation of these proteins is considered an important factor in the assembly of filaments and determination of filament caliber and stability. Mammalian neurofilaments are composed of three polypeptide subunits, NF-L, NF-M, and NF-H, all of which are phosphorylated. Here we used techniques for the mass spectrometric sequencing of proteins from polyacrylamide gels to analyze in vivo phosphorylation sites on NF-M and NF-L. Neurofilaments were isolated from rat brain and enzymatically digested in gel. The resulting peptides were analyzed and sequence data obtained by nanoelectrospray mass spectrometry. Four phosphorylation sites have been found in the C-terminal domain of NF-M: serines 603, 608, 666, and 766. Two of these are found in lysine-serine-proline (KSP) motifs and two in the variant motifs, glutamic acid-serine-proline (ESP) and valine-serine-proline (VSP). Serine 55 in NF-L was not found to be phosphorylated, which confirms the possible role of phosphorylation and dephosphorylation of this site in early neurofilament assembly. The techniques used enable sequence data and characterization of posttranslational modifications to be obtained for each individual subunit directly from polyacrylamide gels.