Table 1.

Troubleshooting guide.

Problem	Possible Cause	Solution
Detection of polymers on FFPE tissue	Wax remaining on tissue	-Use fresh solvents -Increase the number of xylene washes
Poor detection of peptides from fresh frozen tissue vs FFPE tissue	Salts, lipids and metabolites remain on the tissue	-Use fresh Carnoy’s solution each time -Increase the number of washes with Carnoy’s solution
Tissue loss during sample preparation	Antigen retrieval conditions too harsh for tissue type	-Decrease length of antigen retrieval (e.g., fatty and fibrous tissues often reduced to 30 min antigen retrival)
	Slide not adhesive	-Use a positively charged slide -Coat slide with poly-L-lysine
Poor detection of peptides (from either FFPE or FF)	Low humidity during digestion	-Ensure humidity chamber is producing humidity prior to placing the sample in the chamber (Figure 1B)
	Incorrect temperature during digestion	-Check that the oven temperature is 37° ± 1.5°C
	Instrument dirty	-Clean the instrument. A general approach is weekly bakeout of a MALDI FT-ICR that performs high volumes of imaging.
	Laser degradation	-Increase laser power in increments to evaluate impact on signal.
Poor detection of peptides from whole tissue extraction	Low or small amount of tissue	-Perform the preparation on several sections and combine the extracts
Poor detection of peptides from regional locations on tissue	Low amount of peptide due to small region	-Use of nanoESI on a pressure loaded column is recommended -Perform the preparation on several sections and combine extracts
Poor fragmentation of peptides by MALDI – MS/MS	Poor instrument settings	-Use trypsin autolysis peptides to evaluate fragmentation of a target peptide from tissue. -A solution of BSA peptides or a solution of Glufib may also be useful for method setup -Increase the number of scans averaged -Increase collision energy -Increase the isolation window (increases fragmentation of near isobaric peaks)