TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
14	Low number of spectra	Sample of high complexity (samples’ co-elution)	Further fractionate or simplify the sample (i.e., by adjusting the HPLC gradient to make it more gradual to better separate peptides) and thus increase the number of spectra observed
		Insufficient amount of injection or sample	Determine the optimal amount of the sample for the type of MS instrument and LC gradient used. Adjust the amount of the sample loaded. Check the autosampler for possible failure
26	Low-quality tandem mass spectral data sets	Problems with the chromatography column or system	Use a simple protein digest (such as cytochrome c digest) to verify the integrity of the HPLC system
		Insufficient amount of injection or sample	Determine the optimal amount of the sample for the type of MS instrument and LC gradient used. Adjust the amount of the sample loaded. Check the autosampler for possible failure
		Heavy contamination, such as polymers leaching from laboratory plasticware	Use MS-compatible grades of plastic tubes, tips and powder-free gloves as indicated throughout the protocol
	Low ‘spectrum-spectrum match ratio’	Low-quality tandem mass spectral data sets	Open the raw data file and examine the total ion chromatogram by using the mass spectrometer’s software to check the quality of the data set. A good-quality data set should have numerous peaks that rise above the noise in the total ion chromatogram. If the quality is not good, see the troubleshooting section for low-quality tandem mass spectral data sets
		The species of the query sample is not represented in the library	Extend the library data set by adding more species