The in-gel tryptic
digestion was
carried out by following the protocol described previously.44 (link) Briefly, the gel bands were cut into 1 mm3 pieces, rinsed, and dehydrated, and the protein was reduced
with DTT and alkylated with iodoacetamide in the dark prior to overnight
digestion with trypsin at 37 °C in 50 mM ammonium bicarbonate.
The concentrated peptides were analyzed on an LTQ Orbitrap Velos (Thermo
Fisher Scientific, San Jose, CA) coupled with an Eksigent nanoLC-Ultra
1D plus system (Dublin, CA). Peptides were separated on a PicoFrit
analytical column (100 mm long, ID 75 μm, tip i.d. 10 μm,
packed with BetaBasic 5 μm 300 Å particles, New Objective,
Woburn, MA) using a 35 min linear gradient of 5–35% ACN in
0.1% FA at a flow rate of 250 nL/min. Mass analysis was carried out
in data-dependent analysis mode, where MS1 scanned the full MS mass
range from m/z 300 to 2000, at 30 000
mass resolution, and 10 CID MS2 scans were sequentially carried out
in the Orbitrap and the ion trap, respectively.
digestion was
carried out by following the protocol described previously.44 (link) Briefly, the gel bands were cut into 1 mm3 pieces, rinsed, and dehydrated, and the protein was reduced
with DTT and alkylated with iodoacetamide in the dark prior to overnight
digestion with trypsin at 37 °C in 50 mM ammonium bicarbonate.
The concentrated peptides were analyzed on an LTQ Orbitrap Velos (Thermo
Fisher Scientific, San Jose, CA) coupled with an Eksigent nanoLC-Ultra
1D plus system (Dublin, CA). Peptides were separated on a PicoFrit
analytical column (100 mm long, ID 75 μm, tip i.d. 10 μm,
packed with BetaBasic 5 μm 300 Å particles, New Objective,
Woburn, MA) using a 35 min linear gradient of 5–35% ACN in
0.1% FA at a flow rate of 250 nL/min. Mass analysis was carried out
in data-dependent analysis mode, where MS1 scanned the full MS mass
range from m/z 300 to 2000, at 30 000
mass resolution, and 10 CID MS2 scans were sequentially carried out
in the Orbitrap and the ion trap, respectively.