Picotip emitter

Desalted samples reconstituted in 0.1% formic acid were delivered into a QTRAP 6500 (Sciex) via a Dionex U3000 nano-LC system (Thermo) mounted with a NanoAcquity 5μm, 180 μm x 20 mm C₁₈ trap and 1.7 μm, 75 μm X 100 mm analytical column (Waters)maintained at 40°C. A gradient of 2−50% acetonitrile/0.1% formic acid (v/v) over 45 minutes was applied to the columns at a flow rate of 300 nL/minute.The NanoSpray III source of the mass spectrometer was fitted with a 10 μm inner diameter PicoTip emitter (New Objective). The mass spectrometer was operated in positive ion mode using Analyst TF1.6 software (Sciex) and the MIDAS approach (MRM-initiated detection and sequencing) was used to quantify and confirm the identity of the analytes of interest. The optimized transitions are shown in Supplementary Table 1; dwell time for each transition was 20ms. The charge status of each precursor ion was determined using an enhanced resolution scan at 250Da/s and up to 3 MS/MS scans at 10,000Da/s were triggered with dynamic fill time. This gave a total cycle time of 3.9s.

Phosphopeptide-enriched samples were analyzed by LC-MS/MS on an Easy-nLC 1000 (ThermoFisher) coupled to an LTQ-Orbitrap Fusion mass spectrometer (ThermoFisher) operated in positive ion mode. The LC system, configured in a vented format consisted of a fused-silica nanospray needle (PicoTip emitter, 50μm ID × 20cm, New Objective) packed in-house with ReproSil-Pur C18-AQ, 3μm and a trap (IntegraFrit Capillary, 100μm ID × 2 cm, New Objective) containing the same resin as in the analytical column with mobile phases of 0.1% FA in water (A) and 0.1% FA in MeCN (B). The peptide sample was diluted in 20 μL of 0.1% FA, 3% MeCN, and 8.5 μL was loaded onto the column and separated over 210 minutes at a flow rate of 300 nL/min with a gradient from 5 to 7% B for 2 minutes, 7 to 35% B for 150 minutes, 35 to 50% B for 1 minute, hold 50% B for 9 minutes, 50 to 95% B for 2 minutes, hold 95% B for 7 minutes, 95 to 5% B for 1 minute, re-equilibrate at 5% B for 38 minutes. A spray voltage of 2000 V was applied to the nanospray tip. MS/MS analysis occurred over a 3 second cycle time consisting of 1 full scan MS from 350–1500 m/z at resolution 120,000 followed by data dependent MS/MS scans using HCD activation with 27% normalized collision energy of the most abundant ions. Selected ions were dynamically excluded for 45 seconds after a repeat count of 1.

Phosphopeptide enrichment and LC-MS/MS were carried out as described
previously (Juvvadi et al., 2013 (link); Juvvadi et al., 2015 (link); Shwab et al., 2017 (link)). GFP-Trap® affinity
purified protein was processed for TiO₂ phosphopeptide enrichment and
mass spectrometry. Proteolytic digestion was accomplished by the addition of 500
ng sequencing grade trypsin (Promega, Madison, WI) directly to the resin, with
incubation at 37°C for 18 h. Peptides were subjected to phosphopeptide
enrichment using a 10 μl GL Sciences TiO₂ Spin Tip. The dried
phosphopeptide enriched samples were resuspended and subjected to
chromatographic separation on a Waters NanoAquity UPLC equipped with a 1.7
μm HSS T3 C18 75 μm I.D. x250 mm reversed-phase column. The
analytical column was connected to a fused silica PicoTip emitter (New
Objective, Cambridge, MA) with a 10 µm tip orifice.
Phosphopeptide-enriched samples were analyzed on a QExactive Plus mass
spectrometer using a data-dependent mode of acquisition. MS/MS spectra of the 10
most abundant precursor ions were acquired with a CID energy setting of 27 and a
dynamic exclusion of 20 s was employed for previously fragmented precursor
ions.