TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
3	It is difficult to filter the plasma sample	The syringe filter is clogged or the sample concentration is too high	Mix plasma sample with immunodepletion buffer A (plasma/buffer A, 1:5, vol/vol), then centrifuge the sample at 15,000g for 10 min at 4 °C. Filter the supernatants through the syringe filter
6	Efficiency of the immunodepletion is low	The immunodepletion column is deteriorated	Replace with a new immunodepletion column
10	It is difficult to filter the alkylated plasma sample	The sample becomes more viscous after protein alkylation	Thoroughly vortex the alkylated sample and change to a new filter
	Pressure in the 2D HPLC system is building up	The inlet filters and AEX or RP columns may be clogged	Replace the affected inlet filter or AEX/RP column if necessary
	Pressure in the 2D HPLC system is fluctuating, reduced or completely lost	There may be an air bubble in the pump head or a leak in the 2D HPLC system, detached fittings, or a clogged column	Purge the pump to expel air bubbles. Check for loose fittings. Retighten all connections. Check for clogging of inlet filters or columns
	Retention times shift	Changing mobile-phase composition	Replace mobile phases
22	High back pressure in the capillary C18 column	Fragmentation of packing materials	Repack the column
	Unstable spray	Spray voltage set too low or too high	Optimize the spray voltage
35	Few results pass the filtering of glycopeptide probability ≤0.05	Low-abundance glycopeptides can result in noisy spectra	Confirm that there is a low-information content spectrum (i.e., spectrum with few or no signal peaks) by performing Step 33 on some or all spectra without filtering. Reacquire data from other spots in the same well or acquire data from a fresh sample plate
	Expected glycopeptides are not in the results	Glycoprotein ID is not present in refinement protein database	Indistinguishable proteins identified during the refinement database construction should be included when performing Step 23. Protein that is not confidently identified for inclusion in the refinement protein database should be excluded. Include the protein ID when performing Step 25 and repeat the process if you can demonstrate the presence of the protein by other assays
		Glycan is not present in glycan database	Contact author for glycan database update
	Mass spectrum contains a mixture of ions that appears too complex for one glycopeptide	In some cases, co-eluting glycopeptides and peptides may have very similar m/z values and appear in the same isolation window	Check whether at least one of the measured masses in the isolation window matches the calculated mass with the correct isotopic peak with an acceptable delta mass. In this case, the glycopeptide is considered to be a putative true hit
37	The triplet peaks of the X0,2 of N-glycan with the intact peptide backbone are not among the tallest peaks	Insufficient fragmentation	Check if the peaks in the region closer to the precursor have much higher intensities. Reacquire data if this is the case