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.
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.
