TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
7	Decrease in the total number of proteins identified	Issue with depletion or depletion column aging	Run a QC the columns by digesting and analyzing an aliquot of depleted reference plasma sample (e.g. from a commercial source such as BioreclamationIVT) by LC-MS/MS. An Increase in the total intensity of the most abundant proteins targeted for depletion such as albumin and immunoglobulins would indicate degrading columns. See Supplementary Figure 2.
49	The iTRAQ labeling efficiency is < 95%	Insufficient labeling reagent is used	If iTRAQ labeling efficiency is suboptimal, a second reaction with another aliquot of label can be done to improve it. To do that, thaw the original reaction mixture, add 50μL of 100% ethanol and transfer contents of one vial of iTRAQ label solution to reaction mixture. React for 1 h at RT. Repeat the QC analysis.
	The TMT labeling efficiency is < 95%	Insufficient labeling reagent was used	If TMT labeling efficiency is suboptimal, a second reaction with another aliquot of label can be done to improve it. To do that, thaw the original reaction mixture, and transfer contents of one vial of TMT label solution to reaction mixture. React for 1 h at room temperature. Repeat the QC analysis.
56	Loss of resolution and/or broadening peak width of the peptide standards on bRP column	Deteriorating RP column or issue with the HPLC system	Troubleshoot the HPLC system to make sure the pumps, injector and fraction collector are performing optimally. If performance of HPLC system is optimal, test another RP column.
	Increased back pressure on bRP column	Deteriorating RP column or issue with the HPLC system	Troubleshoot the HPLC system to make sure the pumps, injector and fraction collector are performing optimally. If performance of HPLC system is optimal, test another RP column.