Step	Problem	Possible Reason	Solution
10	Low cross-linking yield	Cross-linking experiment failed (Step 2)	Check cross-linking by gel electrophoresis
		Buffers or additives contain primary amines (Step 2)	Change buffer system
		Concentration of cross-linker is too low	Check cross-linking yield by gel electrophoresis and adjust amount of cross-linking reagent
		In-gel digestion failed (Step 3A)	Check digestion by analysis of non-cross-linked peptides; if digestion was not sufficient use higher amounts of trypsin and/or use fresh trypsin stock solution for digestion
		Cross-linked peptides were not extracted from gel (Step 3A)	Try analyzing option 3(B)
		In-solution digestion failed (Step 3B)	Check digestion by the presence of non-cross-linked peptides; if digestion was not sufficient use higher amounts of trypsin and/or use fresh trypsin stock solution for digestion
		Sample loss during SCX (Step 3B)	Check presence of peptides before and after SCX; test the protocol with standard protein (without cross-linking), repeat and strictly follow the protocol
		Problems with LC-MS setup, sensitivity of the mass spectrometer (Steps 4 and 5)	Check LC-MS system using a standard peptide mix; optimize instrument/method parameters; load higher amounts of the sample onto the column, re-calibrate the instrument
10	No cross-links obtained/ low cross-linking yield	Database search parameters not adjusted (Step 9)	Vary database search parameters (e.g. increase precursor and product ion tolerance, lower minimum scores)
11	No peak pairs (cross-links obtained only from BS3-d0 or BS3-d4 cross-linking experiment)	BS3-d0 or BS3-d4 cross-linking experiment failed (Step 2B)	Check cross-linking of the individual reactions without pooling by gel electrophoresis
16	d0/d4 ratios do not show 1:1 ratio	Samples were not pooled in equal amounts	When obtained ratios are shifted, normalize dataset by multiplying ratios with the normalization factor
16	Unreasonable d0/d4 ratios	Generated XICs do not belong to the peak pair (Steps 15-17)	Check elution time window of the cross-linked peptides (BS3-d0 and -d4 cross-linked peptides co-elute)