Table 4.

Troubleshooting guide for profiling the specificity of subtiligase

Problem	Possible Causes	Solutions
Low modification efficiency	pH of peptide library/reaction mixture is too low	Ensure that the pH of the peptide library is close to neutral using pH paper Use a high concentration of tricine in the reaction to maintain pH at 8
No Abu-tagged peptides detected by LC-MS/MS	Many possible causes: Subtiligase variant is inactive TEV protease is inactive Proteome Discoverer search parameters are incorrect	TEV cleave and desalt a sample of the reaction mixture before enrichment to test whether peptides are modified Check Proteome Discoverer search parameters to ensure the Abu mass modification is correct
LC-MS/MS TIC shows evenly spaced repeats	Polymer contamination in the sample	Ensure all de-salting solutions are made using LC-MS grade reagents. Use non-autoclaved plastics.