Table 2.

Troubleshooting the chemical proteomic LC-MS/MS analysis

Issue	Possible Cause	Solution
No protein interface after extraction	Loss of sample after initial extraction Not enough protein used initially Calculated sample concentration incorrectly	Re-use methanol to try to transfer as much protein as possible Prepare larger quantities of cells to ensure you have 1 mg for experiment Double check protein concentration calculated with DC protein assay
Beads in final concentrated sample	Pipetted beads in final elution step	Practice removing eluate from bead solution to verify that you are not removing beads with pipette tip Ensure samples are resting for at least 1 minute after spinning Keep sample tube at the same angle when removing from the centrifuge so that beads do not move around
Extended length peptides	Use of Trypsin instead of Trypsin/Lys-C Trypsin digest was not long enough Trypsin digest was not at the correct temperature	Use Trypsin/Lys-C higher cleavage efficiency lysine residues; use a freshly-aliquoted high-quality enzyme product Digest samples for the full three hours Use a heat block to ensure samples reach correct temperature (37°C) quickly and uniformly