The in vivo state of phosphorylation and the modification of two Cys residu
es of neuromodulin/GAP-43 (Nm) were analyzed by electrospray ionization-mas
s spectrometry (ES-MS). The protein was purified from rat brain with homoge
nization buffer containing 1% Nonidet P-40, protease inhibitors, protein ph
osphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purif
ied by HPLC and ion-exchange chromatography, and the various fractions were
identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetrapho
sphorylated species. All of these Nm species contained 2 mot of added 4-vin
ylpyridine per mot of Nm, suggesting that the two Cys residues are in the r
educed form in the brain. In vivo, the majority of Nm is in the phosphoryla
ted form (similar to 80%), of which the levels of the mono- and diphospho f
orms are higher than those of the tri- and tetraphospho species. Four in vi
vo phosphorylation sites, Ser(41), Thr(95), Ser(142), and Thr(172), were id
entified by amino acid sequencing and tandem ES-MS of the peptides derived
from Lys-C endoproteinase digestion. Among these sites, only Ser(41) is a k
nown target of PKC, whereas the kinases responsible for the phosphorylation
of the other three novel sites are unknown. Hypoxia/ischemia caused a pref
erential dephosphorylation of Ser(41) and Thr(172), whereas Thr(95) is the
least susceptible to dephosphorylation.