Nanoacquity hplc

LC-MS/MS analysis was carried out using a nanoAcquity HPLC (Waters, Milford, MA) and a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). For each analysis, 5 μL of tryptic digest was loaded onto a 180 μm x 20 mm trap column packed with 5 μm Symmetry C18 resin (Waters) using solvent A (0.1% formic acid in Milli-Q H₂O [Millipore, Billerica, MA]) followed by separation on a 75 μm x 250 mm analytical column packed with 1.7 μm BEH130 C18 resin (Waters). Peptides were eluted with either a 4 or 8 hour chromatographic gradient using solvent A and solvent B (0.1% formic acid in acetonitrile). For the 4 hour gradient, peptides were eluted using 5–28% B over 170 minutes, 28–50% B over 50 minutes, 50–80% B over 10 minutes, constant 80% B for 10 minutes, and 80–5% B over 5 minutes. For the 8 hour gradient, peptides were eluted using 5–28% B over 350 minutes, 28–50% B over 150 minutes, 50–80% B over 20 minutes, constant 80% B for 10 minutes, and 80–5% B over 5 minutes. A 30 minute blank gradient was run in between each sample injection to minimize carryover. Full scans were carried out from 400–2000 m/z with 60,000 resolution. MS2 data were acquired in data-dependent mode of the top six most intense ions with dynamic exclusion enabled for 60 s, monoisotopic precursor selection enabled, and single charged ions rejected.

Silver or Coomassie stained SDS-PAGE gel bands were excised and digested in-gel with sequence grade trypsin (Roche, Switzerland). After 5% formic acid/50% acetonitrile extraction, peptides were dried by vacuum centrifugation, gel particulates were removed via C18 Zip Tips (MERCK Millipore, Billercia, MA), dried, resuspended in 0.1% formic acid and analyzed by LC-MS/MS. Chromatography was performed on a Nanoacquity HPLC (Waters, Milford, MA) at 400 nl/min with a BEH130 C18 2.1x150 mm column (Waters). A 90- or 120-minute gradient from 98% solvent A (0.1% formic acid) to 22% solvent B (0.1% formic acid in acetonitrile) was used. Peptides were analyzed by an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Waltham, MA). After the survey scan of m/z 400–1,600 was measured in the Orbitrap at 30,000 resolution, the top three multiply charged ions were selected for both HCD and ETD. Automatic gain control for MS/MS was set to 2000. Normalized collision energy for HCD was set at 35 while the ETD activation time was charge state dependent, based on 100 ms for doubly charge precursors. Supplemental activation was implemented for ETD reactions. Dynamic exclusion of precursor selection was set for 25 s.
WT GlcNAc-S248, WT unmodified and the S248A SOX2 TAD were synthesized by New England Peptide and analyzed as described above.

After proteolytic digestion, the resulting peptide samples were analyzed in a nanoHPLC-MS system consisting of a nanoAcquity HPLC (Waters, USA) equipped with trap (Waters, USA, 180 μm × 20 mm, particle size 5 μm, pore size 120Å, C-18 resin) and analytical (Waters, USA, 75 μm × 250 mm, particle size 1.7 μm, pore size 120Å, C-18 resin) columns, coupled with an Orbitrap Velos mass spectrometer (Thermo Scientific, USA). Peptides were separated with a standard 0–40% gradient of acetonitrile in 0.1% formic acid. The mass spectrometer was operated in data-dependent mode with each MS scan followed by up to 5 MS/MS scans. Both MS and MS/MS spectra were acquired with an Orbitrap detector. Higher-energy Collisional Dissociation (HCD) was used for peptide fragmentation. For MS spectra the resolution of the Orbitrap was set to 30,000 and for MS/MS spectra it was set to 15,000.

Approximately 1 μg of reconstituted peptides, as estimated by a BCA assay (Pierce), was injected into a Waters nanoAcquity HPLC coupled to an electrospray ionization Orbitrap mass spectrometer (QE-HF; Thermo Fisher Scientific; Xcalibur 4.0 software). Peptides were separated at 60 °C on a 100 μm-inner-diameter column packed with 20 cm of 1.7 μm ethylene-bridged hybrid C18 particles (Waters) and eluted at 0.3 μL/min in 0.2% formic acid with a gradient of increasing acetonitrile over 2.5 h. A full mass scan [300−1500 mass/charge ratio (m/z)] was performed in the Orbitrap at 60,000 resolution. The 10 most intense MS1 peaks were selected for fragmentation by higher-energy collisional dissociation at 42% collision energy, and the resulting fragments were analyzed with a resolution of 7500 and an isolation width of 2.5 m/z. Dynamic exclusion was enabled with a repeat count of 2 over 30 s and an exclusion duration of 120 s.