Nanoacquity beh c18 column

Three µg of peptides from each total cilia proteome sample and one third of proximity labeling assay and immunoprecipitation samples were analysed on an LC–MS system composed of an UPLC chromatograph (nanoAcquity, Waters, Milford, MA, USA) directly coupled to a Q Exactive or Elite mass spectrometer (Thermo Scientific, Rockford, IL, USA). Data acquisition for analysis of total cilia proteome was solely performed on Q Exactive system. Peptides were trapped on C18 pre-column (180 µm × 20 mm ,Waters, Milford, MA, USA) using water containing 0.1% FA as a mobile phase and then transferred to a nanoAcquity BEH C18 column (75 µm × 250 mm, 1.7 µm, Waters, Milford, MA, USA) using acetonitrile gradient (0–35% ACN in 160 min) in the presence of 0.1% formic acid at a flow rate of 250 nl/min. Data acquisition was carried out using a data-dependent method with top 12 precursors selected for MS2 analysis after collisional induced fragmentation (CID) with an NCE of 27. Full MS scans covering the mass range of 300–2000 were acquired at a resolution of 70,000 with a maximum injection time of 60 ms and an automatic gain control (AGC) target value of 1e6. MS2 scans were acquired with a maximum injection time of 60 ms and an AGC target value of 5e5 with an isolation window of 3.0 m/z. Dynamic exclusion was set to 30 s.

The
tryptic peptides were analyzed using a Triple TOF 6600 mass spectrometer
(Sciex). Samples were reconstituted in 50 μL of 0.1% formic
acid, and 2 μL of sample was delivered into the instrument using
an Eksigent Nano-LC system mounted with a nanoACQUITY UPLC Symmetry
C18 Trap Column and an analytical BEH C18 nanoACQUITY Column (Waters,
MA, USA). A NanoSpray III source was fitted with a 10 μm inner
diameter PicoTip emitter (New Objective). Samples were loaded in 0.1%
formic acid onto the trap, which was then washed with 2% ACN/0.1%
FA for 10 min at 2 μL/ min before switching in-line with the
analytical column. A gradient of 2–50% (v/v) ACN/0.1% (v/v)
FA over 90 min was applied to the column at a flow rate of 300 nL/min.
Spectra were acquired automatically in positive ion mode using information-dependent
acquisition, using mass ranges of 400–1600 Da in MS and 100–1400
amu in MS/MS. Up to 25 MS/MS spectra were acquired per cycle (approximately
10 Hz) using a threshold of 100 counts per s, with dynamic exclusion
for 12 s and rolling collision energy.

Samples were reconstituted in 2% ACN, 0.1% FA (v/v) prior to analysis using a Triple TOF 6600 mass spectrometer (Sciex, UK) delivered into the instrument using an Eksigent NanoLC Ultra HPLC system. Samples were injected onto a nanoACQUITY UPLC Symmetry C18 Trap Column (P/N Waters, MA, USA) and washed for 10 min at 2 µL/min with 0.1% FA. A gradient from 1.6% ACN/0.1% FA to 95% ACN/0.1% FA was applied over 95 min at a flow rate of 300 nL/min through a Peptide BEH C18 nanoACQUITY Column (Waters, MA, USA). MS was operated as described in previous methods (Meng et al., 2016 (link)).