Steps	Problem	Possible Reason	Solution
Step 9	Incomplete removal of albumin or IgG	Sample exceeds binding capacity	Reduce the amount of sample processed
		Incomplete binding	Increase incubation time
		The sample is not mixed properly during incubation	Mix the sample with resin by gentle end-over-end mixing and make sure that the sample is mixing with the resin during the incubation period
Step 15–17	Incomplete digestion of proteins	pH is not adjusted to ~8	Adjust the pH of the sample by adding 100 mM ammonium bicarbonate.
		Urea concentration can be more than >1 M	Dilute the sample by adding 100 mM ammonium bicarbonate.
Step 18	Too much background	Buffer, salt interference	Clean-up sample with desalting column (Empore C18 disk, Pierce C18, Spin columns)
		Keratin contamination	Use fresh buffer and always wear gloves
Step 40	Less no. of proteins identified in case of iTRAQ/TMT	Less amount of peptide considered for labeling	Label at least 50 μg of each iTRAQ/TMT reagent
		Labeling efficiency is not good	Check the pH before adding iTRAQ/TMT reagents and incubate it 2 h with intermittent vortexing after every 10 min
Steps 40	Chromatogram is not good	LC setting	Optimize LC setting for your sample
		Salt and other contaminants	Clean-up sample with desalting column (Empore C18 disk, Pierce C18, Spin columns)
		Column issue	Replace the column
Step 40	Fragmentation of the peptides is not good	Poor ionization	Clean the front end of the mass spectrometer
		Bubble issue at the tip of column	Increase the voltage till 2.3 kV, if not resolved, change the analytical column