Table 2 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
44	Large amounts of OCT contamination seen in QC mass spectrometry runs High missed-cleavage rates for tryptic peptides (Supplementary Fig. 2)	Large contamination of OCT in cryopulverized tissue Digest efficiency, especially for trypsin, is dependent on enzyme concentration during digestion	Use basic RP fractionation to dilute OCT signal across multiple fractions Use low volumes of extraction buffer to increase the initial protein concentrations
73, 74	Poor labeling efficiency and/or poor mixing control	High levels of plasma contamination, leading to poor labeling efficiency or inaccurate protein BCA assay	If a channel does not have sufficient labeling incorporation, additional TMT is added to the sample and another 1-h incubation is performed with shaking. If the samples are too variable from each other in the mixing control, additional material should be labeled and incorporated, or material should be removed, depending on the nature of the channel, until the channels are ~1:1:1…
97	High-pH separation resolution is <75-80% uniqueness per fraction. For optimal performance across three laboratories and different column types, see Supplementary Fig. 1	Column is overloaded or deteriorated	Use standard runs of synthetic peptides to monitor column performance and do not load more than 4 mg of peptides onto 4.6 × 250-mm columns
126	Phosphopeptide enrichment yields <90% enrichment specificities. For optimal performance across three laboratories, see Supplementary Fig. 4	pH of peptides in IMAC-binding buffer is >2, IMAC beads were not prepared freshly, or IMAC incubation was longer than 30 min	Check pH with filter paper, prepare new IMAC beads for each experiment, and do not incubate for >30 min