Table 2.

Troubleshooting guide for generation of proteome-derived peptide libraries

Problem	Possible Causes	Solutions
Proteins do not dissolve after TCA precipitation	pH is too low Protein solution is too concentrated	Check that the pH is 7.0–8.0 Add buffer to achieve an estimated protein concentration of ≤2 mg/mL
Low number of peptides identified by LC-MS/MS	Incomplete digestion with protease Inefficient de-salting	Check the digested proteins by SDS-PAGE gel to verify no bands above 10 kDa remain or extend protease digestion time. Ensure the pH of the peptide sample is below 3 before apply to C18 and that the resin is equilibrated properly.
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.