Characterization of ADP-ribosyl-HNP-1 from BAL fluid by RP-HPLC, MALDI, and enzymatic digestion. (a) Alignment of RP-HPLC chromatograms of products of ART-1-catalyzed ADP-ribosylation of HNP-1 (continuous line) and a chromatogram of a BAL sample from a smoker (dotted line). Arrows indicate elution times of ADP-ribosyl-HNP-1 and HNP-1. (b) (i) The peak with elution time compatible with ADP-ribosyl-HNP-1 (46.5 min), analyzed by MALDI-MS, shows a mass of 3,983 Da, consistent with ADP-ribosyl-HNP-1 (calculated 3,983 Da). (ii) Incubation of ADP-ribosyl-HNP-1 with pyrophosphatase and alkaline phosphatase produced ribosyl-HNP-1 (calculated 3,574 Da). (iii) ADP-ribosylarginine hydrolase cleaved the ribose-arginine linkage to release HNP-1 (calculated 3,443 Da). m/z is on the x axis.