Table 1.

CEM synthesizer coupling steps

Step	Problem	Possible Reason	Solution
23	Low yield of TFA-NHP	Degraded or contaminated reagents used during synthesis.	Use fresh, high purity reagents. Trifluoroacetic acid anhydride is prone to hydrolysis and should be used within a week of opening.
42	Low yield of cross-linker during resin cleavage, resin has uncleaved cross-linker remaining.	Residual DMF on cross-linker resin inhibits cleavage.	Wash resin extensively with DCM, and then incubate the resin in DCM for 20 minutes, pipetting occasionally to improve solvent accessibility. Make a fresh stock of cleavage solution and repeat the incubation in cleavage solution.
42	Impure cross-linker observed during direct infusion ESI-MS analysis.	Degraded or contaminated reagents used during synthesis.	Make sure all solvents and reagents used for synthesis are freshly made and of high purity. Solvents should be ACS grade or higher. DMF should not be more than 6 months old. Amino acid and DIC solutions are stable for up to two weeks. The piperidine solution is stable for one month.
44	Cross-linker has hydrolyzed.	Cross-linker stock solution has become contaminated with water.	Verify hydrolysis by ESI-MS anlaysis. See Fig. 4.
65	Cross-linker precipitated in pipet tip.	Pipet tip was immersed into aqueous buffer prior to expelling the concentrated cross-linker solution causing it to precipitate	Do not immerse pipet tip in the solution when adding cross-linker. Instead hold the tip above the solution and proceed with pushing the cross-linker solution out.
66	No yellow color observed during cross-linking reaction.	pH of solution is too low.	Check the pH of the reaction solution. It should be between 7–8. If it is lower adjust it within range by titrating with 1M NaOH or use a higher concentration of buffer.
73	Cell pellet is small or solution is sticky and viscous.	Cells have lysed during pelleting and resuspension.	Check centrifugation speed to make sure it is appropriate for the sample type used. Be extremely gental while using a pipet to resuspend cells only using a minimal number of cycles to suspend the cell pellet.
75	Tissue samples not lysing during cryogrinding.	Cryoginding ball was added to liquid sample prior to freezing in liquid nitrogen and is now encased in ice and not able to grind sample.	Add cryogriding ball after sample solution has been frozen in liquid N2 to ensure it is free to move during shaking.
80	Large amount of precipitation in tryptic digest sample.	Typsin failed to digest proteins into peptides.	Sample solution conditions were not compatible with trypsin. Check pH to ensure it is between 7–8. Ensure urea concentration is less than 1 M. Correct solution conditions and add fresh trypsin to repeat step 80.
81	Large amount of precipitation observed after acidifying sample.	Large amount of residual undigested protein due to inefficient trypsin digestion	Check solution conditions for trypsin digestion. Make sure the pH is between 7–8, urea concentration is less than 1 M and fresh trypsin is used. Repeat steps 80–81.
97	No signal or low signal observed in SCX absorbance traces.	Loss of protein during desalting due to poor binding to the packing material.	Check pH of the input sample for the Sep-Pak. Reacidify if above 3, and repeat the desalting procedure.
111	Spray is sputtering or droplets forming at the tip of the column.	Incorrect spray voltage or tip position, blunt, broken or dirty spray tip.	Adjust the applied spray voltage and tip position until stable spray is obtained. If tip appears blunt or dirty replace with a new column.
112	No signal or low signal observed in chromatogram on mass spectrometer.	Poor binding of sample to monomeric avidin resin.	Verify that the pH of the SCX fractions after base addition are approximately 8. These samples can be recaptured again after the pH is corrected.
		Sample not injected by autosampler on LC	Verify that autosampler is delivering the requeseted volume of sample onto the LC column.
		Mass spectrometer is out of calibration or has low sensitivity due to dirty ion optics.	Clean and calibrate mass spectrometer verifying sensitivity with a standard sample.
		Multiple contributing factors.	Use a cross-linked purified protein such as BSA as a positive control sample and ensure that results similar to those presented Fig. 5 and Table 2 are obtained.
120	No significant identifications in .pep.xml file.	Incorrect database or search parameters.	Check that the correct protein database was used and the the comet search parameters match the experimental setup.