Quantitative Nanoflow LC-MS/MS Proteomics

Proteins were analyzed by nanoRPLC–MS/MS. For each TDP LC injection, 6 μL was loaded onto a trapping column (detailed below) with a Dionex Ultimate 3000 RSLC system (Thermo Fisher Scientific, Sunnyvale, CA) and washed with Buffer A for 10 min at 3 μL/min. The 90 min LC gradient used was previously described.^{2 (link)} Autopilot-based identification used PLRP-S media (Agilent, Santa Clara, CA) packed in-house into 2 cm long × 150 μm inner diameter trapping columns coupled to 10 cm long × 75 μm inner diameter analytical columns. Alternatively, the TDQ platform utilized 2 cm Dionex Pepswift trapping columns and monolithic Thermo Dionex RP-4H analytical columns (100 μm ID × 50 cm long) at a flow rate of 1 μL/min and heated to 35 °C. Samples were loaded and washed for 3 min at 10 μL/min. The gradient was the same as for the TDP runs. Electrospray tips were packed with <1 cm of PLRP-S media to reduce bubble formation and promote stable ESI. An Orbitrap Elite (Thermo Fisher Scientific, San Jose, CA) collected all MS data. Xcalibur was the acquisition platform for TDQ quantitative scans where only MS¹ was performed. Autopilot acquired all TDP data. Parameters for Autopilot were as outlined previously.^{8 (link)}