Inject each sample on the in-house assembled nanoPOTS autosampler utilizing an in-house packed SPE column (100 μm i.d., 4 cm, 5 μm, 300 Å C18 material,Phenomenex) for an online sample clean up, and an LC column (50 μm i.d., 25 cm,1.7 μm, 190 Å C18 material, Waters) which is heated to 50 °C using an AgileSleeve column heater (Analytical Sales and services, Inc., Flanders, NJ) for the peptide chromatographic separations. Dissolve samples with Buffer A [0.1% formic acid in water] on the chip, then load peptides on the SPE column and wash the trapped peptides with Buffer A for 5 min. After sample cleaning, separate and elute the peptides at 100 nL/min using a 60-min gradient, linearly increasing Buffer B [0.1% formic acid in acetonitrile] from 8% to 35%