Table 2. Troubleshooting table. [As far as possible, please include the step number(s) at which the problem first becomes evident.].

Step	Problem	Possible Reason	Solution
1	Protein precipitates either on-the column or in solution	Incorrect pH	The pH is not ideal for sample. Try using Mem MS Buffer at pH 8.0, or other pH values.
		Incompatible detergent	The membrane protein is not soluble in this detergent. Choose a different MS-compatible detergent from table 1.
9	Intermittent spray or fluctuating ion current	Capillary blocked or narrow tip diameter	Trim back the capillary tip with tweezers.
		Protein too concentrated	High protein concentrations tend to form viscous solutions making it difficult for nanospray. Dilute the sample in Mem MS Buffer.
		Tip incorrectly trimmed	Ensure the capillary tip is smooth and not jagged. If so, either trim back the tip with tweezers or smoothen by pushing the tweezers against the tip.
9	Loss of spray or ion current	Sample aggregated	See problem ‘Protein crashed out…’ above.
		Gold coating stripped off the tip of the capillary	Trim back the capillary tip with tweezers.
		Cone covered in detergent solution	Remove and clean the cone.
10	Broad peak(s) and/or excessive adducts as seen in mass spectrum	Sample contains impurities and/or excess detergent, salts, and/or lipids	Prepare membrane protein sample by gel filtration to increase purity and/or remove adducts (step 1B). If problem remain try a detergent screen (BOX 2).
10	Ion series offset by regular interval	High concentration of detergent in sample leading to mass spectra of detergent micelles.	Dilute sample into Mem MS Buffer in the absence of detergent to dilute detergent.
10	Charge state series is complicated	Detergent choice affecting ionization and/or producing detergent clusters	Find another detergent using the detergent screen described in BOX 2. Alter the activation energy to remove detergent from the membrane protein complex and to disrupt detergent clusters (see Figure 3)
13	No lipid bound during lipid titration	Chloroform present in solution	Prepare lipid stocks as described in BOX 3 with added care and patience in organics removal step
		Low concentration of lipid to protein	Perform higher titration series. Try another lipid type; lipid binding will be protein dependent with potential for no lipid binding.
		Detergent used is not competed off by lipid	Perform detergent screen as described in BOX 2 and attempt lipid binding studies thereafter.
13	Broad hump seen during lipid binding studies	High concentration of lipid to protein	Perform titration series at a lower concentration of lipids.