MAIDI-MS analysis of HPLC fractions with major radioactivity.

1The wild-type hTTP protein was purified from transfected human cells after in vivo radiolabeling with [32P]-orthophosphate. The protein was purified and digested by trypsin to completion. The phosphopeptides were identified by radioactivity peak on HPLC chromatogram (Figure 6A).2The observed peptide mass of [M+H] ion was obtained after phosphopeptides were sequenced by MAIDI-MS.3The unmodified peptide mass of [M+H] ion was obtained after theoretical digestion of His-hTTP with trypsin.4The differential mass was obtained by subtraction the unmodified ion mass from the observed ion pass. Phosphorylation results in a peptide ion with a +80 Da mass increase compared to the unmodified peptide for each phosphorylated Ser, Thr or Tyr residue (HPO3− = 79.97 Da).