TABLE 2.

Troubleshooting table.

Problem	Possible reason	Solution
No high molecular weight proteins after cell lysis	Protein degradation	Make sure protease inhibitors were added before lysis. Avoid sample heating during lysis. Perform lysis steps up to protein digestion (Steps 1–7) the same day and as fast as possible
No peptides observed in checkpoint 2 (Box 1)	Protein digestion failed	Check integrity/reactivity of trypsin solutions. pH during digestions should be 7.5–8.5 and urea concentration <2 M
Peptides elute too late when analyzed by LC-MS/MS	Maximum capacity was reached on the SepPak cartridge used for peptide desalting	Use higher-capacity SepPak cartridge or save the flow-through and washes and perform a second desalting step with them
Peptides elute too early in the SCX. Peptides with high solution charge are observed in the early fractions	SCX column has not been equilibrated properly	Equilibrate the SCX column for a longer time
	SCX buffers A and B have pH > 3	Make new buffers and ensure they have been properly prepared
	SCX buffer A has higher concentration of cations
Many nonphosphorylated peptides (>50%) are detected after IMAC enrichment	Too much IMAC resin was used for the amount of peptides	Reduce the amount of resin
	Incubation time of peptides with IMAC resin was too long	Do not incubate for longer than 60 min
	IMAC resin was not properly washed before elution	Add an extra wash step with IMAC-binding buffer (i.e., repeat Step 34A(ii) or 34B(ii))
Very few total peptides were detected	Starting amounts of protein were not sufficient	Although lower amounts might work, we recommend starting with at least 5 mg of protein
	IMAC resin is too old	PhosSelect IMAC resin shelf life is less than 1 year. Store in small aliquots at −20 °C and do not freeze and thaw multiple times. When performance is decreased, replace it with a new batch (an SCX fraction prepared following this protocol can be used as a control)
	There was a problem in the LC-MS/MS system	Make sure LC-MS/MS system is working properly with a complex mixture standard (see EQUIPMENT SETUP)