TABLE 1|.

Troubleshooting table.

Steps	Problem	Possible reason	Solution
Box 1, steps 4, 6–9	Liquid is not spraying from the analytical column	Column is not fully open	Examine the column under a microscope; if it is not fully open, etch the tip by dipping it in HF for 1–2 min (see Box 1 for details. HF is extremely dangerous; follow the precautions outlined in Box 1). The column should also be examined for any obstructions (dust, particles) that may prevent it from packing
		Open end of the column is clogged	The end of the column can become damaged or clogged when inserting it through the ferrule, leading to the high-pressure bomb. After insertion, cut ~0.2 cm off the end of the column using a ceramic scoring wafer
Box 1, step 15	Column is packing too quickly with 3.5-|im packing material	Slurry of 3.5-μm packing material is too concentrated	Make a more dilute solution of packing material by increasing the amount of ACN
Box 1, step 17	Column is not packing		Turn helium off, and then back on, using the valve connected to the pressure bomb. This will sometimes cause the column to begin packing again
		Open end of the column is clogged	The end of the column can become clogged with packing material. Cut ~0.2 cm off the end of the column using a ceramic scoring wafer
		Slurry of 1.7-μm packing material is too concentrated	Ensure that all of the packing material is able to go into solution when stirred; if not, increase the amount of ACN in the slurry (note that ACN will evaporate over time)
		Open end of the column does not reach the packing material slurry	Ensure that the end of the column is fully submerged in the packing material slurry
	Gaps in packing material	Poor flow of packing material; blockage	Increase the pressure of the helium
			Connect to UPLC and flow the 100% mobile phase B through the column at a flow rate of 0.3 μl/min for 15 min. Discard the column if the gap does not fill
Reversed-phase Chromatography (Equipment Setup)	High back-pressure during LC-MS/MS separation	Analytical column may be clogged	Replace the column
		High-pressure union is clogged	Over time, the high-pressure union can become blocked with packing material or fused silica. Remove the union. When held up to the light, you should be able to see through it. If the union is blocked, wash it with ethanol to remove the debris. If necessary, replace the union
		Analytical column is attached too tightly at the union	If the analytical column is attached too tightly, the end can become crushed and block the union. Remove the column and trim the end with a scoring wafer. To avoid this problem, after threading the column through the ferrule, trim the end with a ceramic scoring wafer. The column should protrude ~1 mm out of the ferrule. Tighten the analytical column as much as possible, without crushing the tubing. Once the analytical column is connected, gently pull on it. If it comes loose, use slightly more force when you are tightening the union
		25-μm line is clogged	Verify by removing the analytical column; if there is not a drastic decrease in pressure, replace the 25-μm line
	High back-pressure during sample loading (>8,500 psi)	Impurities in the sample	Ensure that the sample was properly desalted before analysis
Chromatography	Very low back-pressure	Column not properly attached	Verify whether the liquid is flowing from the tip; if not, make sure that the 25-μm line and the analytical column are properly connected at the union. If no liquid flow is observed from the 25-μm line, check its connection to the LC system
	Broad peaks (>1 min)	Column was packed >1 cm with 3.5-μm packing material	Discard the column (see step 11 from Box 1)
		Gaps in packing material in column	Flow 100% solvent A through the column at 300–350 μl/min (with column heater). Inspect the column; if there are no more gaps and the packed column is of suitable length, trim the unpacked end from the column using a ceramic scoring wafer. If gaps persist, discard the column
		Sample overloading	Reduce the amount of material injected on the column
	Late-eluting sample	Column degradation	Inject 100–300 fmol of tryptic BSA peptides on the column; if peaks have shifted significantly, discard the column. Similarly, if total ion chromatogram (TIC) and base peak intensities are significantly lower than usual and the instrument is working as expected, discard the column
		Trapping sample for too long	Decrease the trapping time according to the instructions in the ‘Reversed-phase chromatography’ section (Equipment Setup)
		Column is not packed all the way to the end	Inspect the column using a light source. If it is not packed to the end, remove the unpacked portion using a ceramic scoring wafer
		25-μm line too long	If possible, trim this line to <50 cm
		Pumps are not delivering the correct amount of solvent	Measure the flow rate of pumps A and B; if they are not delivering the correct amount of solvent, the gradient may need to be adjusted to accommodate the actual flow rates. Contact the LC vendor for maintenance help
	Run-to-run variation	Column is not fully equilibrated	Increase the time of the equilibration period at the end of the LC gradient
		Pumps are not delivering the correct amount of solvent	Measure the flow rate of pumps A and B; if they are not delivering the correct amount of solvent, the gradient may need to be adjusted to accommodate the actual flow rates. Contact the LC vendor for maintenance help
		Column degradation	Inspect the chromatogram for wide peaks. This can also be tested by injecting 100–300 fmol of tryptic BSA peptides on the column. If peaks are wider than ~40 s, discard the column
8	Amidation reaction	Urea degradation during reduction (Step 7)	Perform the reduction step (Step 7) at a lower temperature (i.e., at ambient temperature)
24	Fewer identifications than expected; data-base search gives more semi-tryptic peptides than tryptic peptides	Active endogenous proteases	Collect yeast at an earlier time point. If this is not an option, try digesting with a higher enzyme: protein ratio for a shorter period of time (for example, 1:5 ratio for 4 h) (Steps 11,12)
24	Large number (>50%) missed cleavages		Increase the enzyme to protein ratio to 1:10 (Steps 11,12)
14	Protein precipitates out of solution on acidification with TFA		Use a less concentrated solution of TFA; add TFA slowly, by checking the pH after each addition to avoid overacidification
24	Fewer identifications than expected	Instrument is out of calibration	Follow the recommended calibration schedule provided in the calibration console. If MS mass error is >6 p.p.m., recalibrate
	Fewer identifications than expected; decrease in TIC intensity over time	Quadrupole may require maintenance	Over time, the quadrupole and other parts of the instrument may require cleaning or maintenance. Contact your service engineer for instructions