Nano-RPLC Protein Identification Workflow

The nano-RPLC (reverse-phase liquid chromatography) system consisted of a Spark Endurance autosampler (Emmen, Holland) and an ultrahigh pressure Eksigent (Dublin, CA) Nano-2D Ultra capillary/nano-LC system. Mobile phases A and B were 0.1% formic acid in 2% acetonitrile and 0.1% formic acid in 88% acetonitrile, respectively. Four µL of samples was loaded onto a reversed-phase trap (300 µm ID × 1 cm) with 1% mobile phase B at a flow rate of 10 µL/min, and the trap was washed for 3 min. A series of nanoflow gradients (flow rate at 250 nL/min) was used to back-flush the trapped samples onto the nano-LC column (75 µm ID × 75 cm) for separation. The nano-LC column was heated at 52 °C to improve both chromatographic resolution and reproducibility. An LTQ/ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA) was used for protein identification. The parameters for MS are shown in our previous publications.^{4 (link),13 (link)–15 (link)}