Table 1. Protein Preparation Steps for Array of Lysis Buffers Used in SP3 and In-Solution Digestion.

protein solubilization system	collection method	collection buffer	protein recovery and reduction	protein alkylation
6 M urea	Buffer	20 μL 6 M urea, 0.1 M tris	Add DTT to 5 mM; Incubate 30 min, RT	Add IAA to 20 mM; 30 min, RT
	Cap	–	Add 10 μL 6 M urea, 0.1 M tris, 5 mM DTT; Incubate 30 min, RT
6 M urea, 30% acetonitrile	Buffer	20 μL 6 M urea, 100 mM Tris, 30% acetonitrile (v/v)	Add DTT to 5 mM; Incubate 30 min, RT
	Cap	–	Add 10 μL 6 M urea, 0.1 M tris, 5 mM DTT, 30% acetonitrile (v/v); Incubate 30 min, RT
50% Trifluoroethanol	Buffer	20 μL Ultrapure water	Add 20 μL trifluoroethanol. Add DTT to 5 mM; Incubate 60 min, 60 °C
	Cap	–	Add 10 μL 50% trifluoroethanol (v/v), 0.1 M tris, 5 mM DTT; Incubate 60 min, 60 °C
RIPA	Buffer	20 μL RIPA	Add DTT to 5 mM; Incubate 30 min, RT
	Cap	–	Add 10 μL RIPA (Pierce #89900), 5 mM DTT; Incubate 30 min, RT