6520 esi q tof mass spectrometer

For the remainder of this manuscript, solvent A will refer to water with 0.1% (v/v) TFA, while B will be acetonitrile with 0.1% (v/v) TFA. TIC refers to total ion current in the LCMS chromatogram. The majority of LCMS chromatograms and mass spectra were obtained using an Agilent 6520 ESI-Q-TOF mass spectrometer using method 1 unless otherwise noted (MS/MS analysis was conducted on an Agilent 6550 iFunnel Q-TOF mass spectrometer). Software used for LCMS analysis was the Agilent MassHunter package and deconvolution was performed using maximum entropy.

Method 1:

LC method: 0–2 minutes 5% B, 2–11 minutes 5–65% B linear ramp, 11–12 minutes 65% B, 0.8mL/min flow rate.

Column: Zorbax 300SB C₃ column (2.1 × 150mm, 5µm), 40°C

MS parameters: positive electrospray ionization (ESI).

Method 2:

LC method: 0–3 minutes 5% B, 3–17 minutes 5–95% B linear ramp, 17–18 minutes 95% B, 0.8mL/min flow rate.

Column: Zorbax 300SB C₁₈ column (2.1 × 150mm, 5µm), 40°C

MS parameters: positive ESI

Method 3:

LC method: 0–3 minutes 5% B, 3–15 minutes 5–80% B linear ramp, 15–16 minutes 80% B, 0.8mL/min flow rate.

Column: Zorbax 300SB C₁₈ column (2.1 × 150mm, 5µm), 40°C

MS parameters: positive ESI, MS off at 11 minutes

Peptides corresponding to Switch I sequence of Ras (FVDEYDPTIE), Rap1 (FVEKYDPTIE), Ral (FVEDYEPTKA) and Ras Switch I Y32A/D33A (FVDEAAPTIE) were synthesized by fast flow Fmoc solid phase peptide synthesis on H-Rink Amide-ChemMatrix resin^{41 (link)}. Each peptide was purified using an Agilent Zorbax 300SB-C₁₈ column (5 μm, 9.4 x 250mm) at 7 mL/min and 1–31% acetonitrile (0.1% TFA) over 60 minutes. 10 μM of each peptide was then incubated in the presence or absence of RRSP (10 μM) in 20 mM Tris pH 7.5 with 150 mM NaCl in a 37°C water bath. Time points were pulled at 0, 1, 2, 5, and 8 hours, quenched with 1:1 water (0.1% TFA):acetonitrile (0.1%TFA), and analyzed by Agilent 6520 ESI-Q-TOF mass spectrometer with an Agilent Zorbax 300SB-C₃ column (5 μm, 2.1 x 150 mm) at 0.8 mL/min and 1–41% acetonitrile (0.1% formic acid) over 8 minutes.