Graduate Thesis Or Dissertation
 

Mass spectrometric analysis of bovine neurofilament proteins NF-L, NF-M, and NF-H : peptide mapping, phosphorylation and alkylation site identification

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  • Neurofilament proteins are intermediate filaments found in the neuronal cytoskeleton. Phosphorylation of these proteins is considered important for the assembly and stability of the filaments. Accurate molecular weights have been difficult to measure, largely because the high degree of phosphorylation results in M[subscript r]'S that are significantly greater than dictated by their putative sequences. Mass spectrometry has now been used to measure the molecular weights of all three bovine neurofilament proteins, NF-L, NF-M and NF-H, which are 62 kDa, 105 kDa and 125 kDa, respectively. Peptide mapping resulted in the elucidation of many phosphorylation sites in NF-L and NF-M. Sixteen serines and four threonines within the C-terminal tail domain of NF-M were found to be phosphorylated. Ten of these are within the lysineserine- proline (KSP) motif, and two are in the variant motif, glutamic acid-serine-proline (ESP). In addition six phosphorylation sites, Ser-136, 163, 241, 242, and Thr-139, and 184 were identified in the rod domain of NF-M. Phosphorylation sites identified in NF-L include four serines in the head domain, and one serine in the C-terminal domain. Digests analyzed by LC-ESI mass spectrometry combined with database searching resulted in 88.5% sequence coverage of NF-M, 79.2% of NF-L and 38.4% of NF-H. Alkylation of NF-L, NF-M, and NF-H using a known neurotoxin, 2,5-hexanedione resulted in complicated spectra due to crosslinked peptides. Presently, software limitations have prevented complete identification of these peptides or alkylation products.
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