AQUA quantitation. The enzymatic reaction with 2 mM Ub, 1.5 uM E1, 10 uM UbcH7, and 1 uM NleL was analyzed using AQUA. In-gel digestion of the di-Ub band was performed using TrypsinGold according to the standard AQUA protocol. Orbitrap Fusion and Easy-nLC 1000 (Thermo Inc., San Jose, CA) operating in FT full scan MS mode in the 300-1500 Th range was used for LC-MS analysis. Linear response curves were constructed for K-ε-GG tryptic AQUA peptides (cell signaling technologies). Self-packed UHPLC RP column was run with water:acetonitrile mixtures in the presence of 0.1% formic acid using the following gradient: : 0-10% B 5 min, 10-30% B 40 min, 30-50% B 15 min, 50-95% 3 min, 95% hold 7 min. XIC chromatograms were extracted using QualBrowser (Thermo Inc., San Jose, CA) and then exported to OriginPro for calculation of peak areas.