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 resin41 (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.
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Ras GTPase Peptide Synthesis and Mass Spectrometry Assay
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Corresponding Organization : Northwestern University
Other organizations : Massachusetts Institute of Technology
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Variable analysis
independent variables
- Presence or absence of RRSP (10 μM)
- Peptide cleavage or modification over time (0, 1, 2, 5, 8 hours)
- Incubation temperature (37°C)
- Buffer composition (20 mM Tris pH 7.5, 150 mM NaCl)
- Peptide concentration (10 μM)
- Positive control: Incubation of peptides with RRSP (10 μM)
- Negative control: Incubation of peptides without RRSP
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