Waters nanoacquity

Splitless, nano-flow liquid chromatography separation was performed using a Waters nanoACQUITY (Waters Corp., Milford, MA) UPLC system configured for partial loop injection coupled online to the Q-Exactive mass spectrometer. Fused silica capillary tubing (75 μm I.D.; Polymicro Technologies) was pulled to a tip of ~ 5 μm at one end and packed with 15 cm of Jupiter Proteo 4 μm C12 beads (Phenomenex, Torrance, CA). A Kasil fritted trap was prepared using a 100-μm I.D. capillary tube packed with 2 cm of the identical C12 packing material, and 2 μL of sample was trapped at a flow rate of 1 μL/min for 8 min prior to going in-line with the packed tip. Peptides were separated over a 60-min linear reversed phase gradient ranging from 2 to 32% Buffer B (0.1% w/v formic acid in acetonitrile) mixed with Buffer A (0.1% w/v formic acid in water) with a flow rate of 300 nL/min. Quality control runs analyzed a six bovine protein digest (Bruker-Michrom, Auburn, CA) using a 30-min linear gradient.

MS sample preparation and analyses were performed by the Stanford University Mass Spectrometry facility. In brief, for gel-free MS analysis, the final elutions from the immunoprecipitations were solubilized and digested using the filter-aided sample preparation (FASP) protocol [70 (link)]. Trypsin/Lys-C Mix (Promega, Madison, WI, USA) was used for protein digestion. Peptides were extracted and dried using a speed-vac prior to reconstitution and analysis. Nano reverse-phase HPLC was performed using either an Eksigent 2D nanoLC (Eksigent, Dublin, CA, USA) or Waters nanoAcquity (Waters, Milford, MA, USA) HPLC system with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile. A fused silica column self-packed with Duragel C18 (Peeke, Redwood City, CA, USA) matrix was used with a linear gradient from 2% B to 40% B at a flow rate of 600 nL/minute. The nanoHPLC was interfaced with a Bruker/Michrom Advance Captive spray source for nanoESI into either an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Fremont, CA, USA) or an Orbitrap Elite (Thermo Fisher Scientific) operating in data-dependent acquisition mode to perform MS/MS on the top twelve most intense multiply charged cations.

UPLC-QToF nanospray MS (Waters nanoAcquity, QToF Micro) was used to evaluate the high-resolution mass spectrometry. To prepare the UPLC column, solvents A (90% H₂O—AcN, 0.1% FA) and B (60%–H₂O, 0.1% FA ) were added over 75 min with a flow rate of 0.3 µL/min to Water ACQUITY UPLC M-Class Peptide BEH C18 column (1.7 μm, 130 Å, 75 μm × 150 mm)^{73 (link)}.