Tryptic phosphopeptides of AdIGFBP-5, [γ-32P]ATP-labeled in vitro by phosphorylation with CK2, were separated by HPLC and detected and sequenced by mass.

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binding sites 58 of the 473 unambiguously assigned phosphorylation sites are predicted by Scansite to be sites for binding. 50 of these correspond.

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Presentation transcript:

Tryptic phosphopeptides of AdIGFBP-5, [γ-32P]ATP-labeled in vitro by phosphorylation with CK2, were separated by HPLC and detected and sequenced by mass spectrometry.a, [γ-32P]ATP-labeled phosphopeptides were collected in fractions, and the radiation was measured as shown on the left axis. Tryptic phosphopeptides of AdIGFBP-5, [γ-32P]ATP-labeled in vitro by phosphorylation with CK2, were separated by HPLC and detected and sequenced by mass spectrometry.a, [γ-32P]ATP-labeled phosphopeptides were collected in fractions, and the radiation was measured as shown on the left axis. The percent increase in concentration of organic phase (Phase B) is shown on the right axis. Phosphopeptides that matched to the IGFBP-5 sequence are shown for each fraction in which they were detected. b, tandem MS spectrum of the triply charged parent ion at m/z 850.3 from fraction 18 of the HPLC separation shown in a. The sequence matched IGFBP-5-(95–115) where Thr103 is phosphorylated and Met107 is oxidized (shown underlined). The parent ion has been truncated, and the m/z range from 980 to 1,700 has been multiplied by a factor of 15 to show the fragment ions more clearly. c, tandem MS spectrum of the triply charged parent ion at m/z 881.0 from fraction 33. The sequence matched IGFBP-5-(231–252) where Ser249 is phosphorylated. Cys243 was alkylated with acrylamide (see “Experimental Procedures”) and is shown underlined. The y4 and the b4 ions were equal in molecular mass; however, this did not affect identification of the phosphorylation site. Mark E. Graham et al. Mol Cell Proteomics 2007;6:1392-1405 © 2007 The American Society for Biochemistry and Molecular Biology