Modification of CXCL10 by peptidylarginine deiminase (PAD) and RP-HPLC purification of citrullinated CXCL10. Recombinant CXCL10 (100 pmol) was incubated with rabbit PAD (A) or human PAD2 (B) at an enzyme-substrate molar ratio (E/S) of 1:20 or 1:200 for different time periods. Samples were applied on polyvinylidene difluoride membranes for Edman degradation and in parallel were desalted on a C4 ZipTip before examination on an ion trap mass spectrometer to determine the presence of Arg (♦) or Cit (□) at position 5. The percentage conversion of Arg₅ into Cit₅ and the conservation of Arg₈ in the sequence were calculated from the amount of PTH-Arg and PTH-Cit that were detected by RP-HPLC after 5 and 8 cycles of Edman degradation. (C) Recombinant CXCL10 was incubated with rabbit PAD for 90 minutes at an enzyme-substrate molar ratio of 1:20, purified by C8 RP-HPLC, eluted in an acetonitrile gradient, and detected at 214 nm (mAU). Part of the column effluent (0.67%) was split on-line to an ion trap mass spectrometer, and the averaged spectra for the chromatographic peaks were deconvoluted to obtain the M_r of the proteins (inset indicated by ↖).