Table 1.

Overview of the sample preparation and digestion conditions for the basic in‐gel digestion (Method1) and updated protocols (methods 2–6)

Protocol step	Conditions	Method
		1	2	3	4	5	6
Distain	Gel distain	✓	✓	✓	✓	✓	✓
	50% and 100% ethanol
Reduction	10 mM DTT	✓	✓
and alkylation	56 °C, 30 min
	55 mM IAA	✓
	21 °C, 20 min
	55 mM CAA		✓
	21 °C, 20 min
	10 mM TCEP and 40 mM CAA			✓
	21 °C, 20 min
	10 mM TCEP and 40 mM CAA				✓	✓	✓
	70 °C, 5 min
Clean up	Gel wash	✓	✓	✓	✓	✓	✓
	50% and 100% ethanol
Tryptic digestion	Tryptic digestion in 50 mM ABC	✓	✓	✓	✓
	37 °C, over night
	Tryptic digestion in 50 mM ABC					✓
	37 °C, 4 h
	Tryptic digestion in 50 mM HEPES						✓
	37 °C, 4 h
Peptide extraction	Peptide extraction,	✓	✓	✓	✓	✓	✓
	25% and 100% ACN