Table 2 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
35	Different volumes in tubes after centrifugation	Evaporation during ultracentrifugation	Test all buckets with test gradients without lysate, marking the level of liquid in the tubes. Avoid using buckets for which evaporation is observed.
	Variability in number of fractions recovered	Differences between pipet sets or users	Try to use always the same pipet set for every replicate. The replicates should also be performed by the same user.
42	Difficulty to detect protein bands	Less than 1 mg lysate was loaded onto gradient; low sensitivity of antibody; low expression of protein	Load 1–2 mg lysate onto gradient; try an alternative antibody; test control proteins to verify western blot procedure; check protein expression in lysate.
49	Graph is inverted	The ImageJ software gave back inverted mean values	Subtract the inverted mean from 255 (for 8-bit images).
	Negative protein amounts	Excel mixed up decimal separator “comma” and “point”	Check all mean values again; values cannot be higher than 255 (for 8-bit images).
88	TMT label incorporation is <95%	Poor labeling efficiency	Repeat labeling (steps 76–82) for all channels: add another aliquot of TMT reagent to each sample for 1 h and proceed with testing and quenching as described (Steps 83–88).
95	No proteins / peptides left	Pellet got lost in one of the TCA washing steps	Tubes should be placed into the centrifuge always with the same orientation so that it is clear where the pellet is located.
	Low protein / peptide concentration	Warm acetone was used for washing steps during TCA precipitation; pellets were overdried and could not be dissolved in the next steps	Acetone needs to be cold to prevent washing away proteins; decrease time for drying, or incubation in an ultrasonic bath with ice can help to re-dissolve the pellet.