Fig. 4.

MALDI-MS analysis of human MTHFR peptides limits possible phosphorylation sites. The amino acid sequence of histidine-tagged human MTHFR is shown. Digestions of human MTHFR were performed by using trypsin, Lys-C, or Glu-C. Regions of the protein that were not recovered are highlighted in yellow. Regions of the protein that were recovered constitute 88% of the sequence, and no modifications other than N-terminal acetylation were identified. Arrows indicate the start and end of the catalytic domain based on the crystal structure of E. coli MTHFR (24). The boxed sequence KRREED is thought to be the most sensitive position for initial tryptic cleavage of native enzyme, which separates the catalytic and regulatory domains. After extensive digestion of the native enzyme with trypsin, N-terminal amino acid analysis indicated sequences ^*GNSSL and ^*SKSPK, respectively.