TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
5	Low yield of protein or peptide immobilization	The buffer contains amine groups, because a high concentration of amine-containing buffer was used before buffer exchange	Redo buffer exchange to reduce amine groups, minimizing amine reaction with aldehyde resin
7A(iii)	Low solubility of peptides	Peptides are hydrophobic	Add ACN (10% in final solution; e.g., 10 μl in 90 μl of binding buffer) to dissolve the peptides if the binding buffer does not completely dissolve them
15	The resin aggregates after samples are added	The sample cannot interact with the surface of the resin	Use a pipette tip to disrupt aggregation; vortex and/or sonicate the resin for uniform suspension
17	Low immobilization of proteins	An insufficient amount of resin was used, the conjugation time was too short or the pH was too low (<7)	The maximum capacity of the resin is 20 mg/ml; increase the amount of resin, prolong the reaction time or adjust pH up to 10
24A(ii)	It is difficult to adjust pH	Some EDC is very viscous, even at elevated temperatures. This makes it difficult to add the exact amount of EDC to the solution	Use EDC from Sigma-Aldrich (cat. no. 39391). This EDC has low viscosity even at −20°C
24A(v)	EDC-pT remains bound to the resin	The resin was washed or vortexed insufficiently	Add additional washing steps for each solution; vortex the resin in solution for up to 1 min
44	No N-glycans were identified	The sample contains salts or the aldehydes on the resin have not been blocked effectively	Use a PGC ziptip for glycan cleanup; if the resin has active sites on aldehydes, reblock the resin before PNGase F digestion
58	No O-glycans were detected	The pH of the solution was too high, so that O-glycan was dissolved in chloroform instead of in water	Check the pH of the resuspended O-glycan sample; if the pH is high (>7), use 100% TFA to lower the pH (<3)
70	An error occurred when selecting ‘run’	The file directory was incorrect	Make sure that the right file directory is used for each Python command
71	The glycan structure cannot be determined by MS/MS	An insufficient number of MS/MS fragments were provided	In GlycoWorkBench, search for glycan structures with known modifications, based on accurate precursor m/z values. List the potential glycans from the database in GlycoWorkBench