Fig. 1

Tat phosphorylation in cultured cells. a Purification of Flag-tagged HIV-1 Tat for mass spectrometry analysis. Flag-tagged Tat was expressed in 293T cells, immunoprecipitated from cellular lysate with anti-Flag antibodies and resolved on 10% SDS-PAGE. Peptides were in-gel digested with trypsin, eluted and subjected to MS analysis on Thermo LTQ Orbitrap XL mass spectrometer. Position of Flag-Tat is shown. b MS/MS analysis of Tat phosphorylation. SEQUEST search results are shown for Tat peptides identified with high, median and low confidence indicated in green, blue and red, respectively. Peptides that were not detected are shown in black. Phosphorylated serine and threonine residues are marked by asterisks. Identified phosphopeptides are shown in the table. c–f Phosphopeptides spectra. c MS/MS spectra of the Ser-16 phosphorylated Tat peptide 8–19. d Ser-46/Tyr-47 phosphorylated Tat peptide 41–50. e Ser-81 phosphorylated Tat peptide 72–89. f Ser-87 phosphorylated Tat peptide 72–89. The colored peaks indicate matched MS/MS fragments. Green color indicates precursor ions. Blue and red colors indicate y and b ions, respectively