- HDX Mobile phase B: 0.3% (v/v) formic acid in acetonitrile:
H 2 O (ratio of 4:1). - HDX Mobile phase C (protease column buffer): 0.1% (v/v)
TFA in H 2 O. - Protease column: Pepsin immobilized on resin
(1 mm20 mm). - Chromatography columns: 1 mm 10 mm C8 trap and
1mm50 mm C18 analytical columns. - Acetonitrile: Stock solution of 100% (v/v) acetonitrile (ACN).
- Formic acid: 0.3% (v/v) formic acid (FA).
- Mass spectrometer for peptide identification experiments:
LTQ XL™ linear ion trap mass spectrometer or a similar
instrument (seeNote 9). - Instrumentation for differential HDX experiments: Ideally use
a fully automated system, e.g., LEAP Technologies Twin HTS
PAL liquid handling robot interfaced to a high-resolution
(70,000 at m/z 400) Exactive Orbitrap mass spectrometer
(seeNote 10). - Software for analysis of mass spectrometric data: e.g., Mascot
(Matrix Science, London, UK) for peptide identification and
HDX Workbench or similar for differential HDX data
processing.
2.3 X-Ray
Crystallography
- X-ray size exclusion chromatography (SEC) buffer: 25 mM
Tris–HCl, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol,
2 mM TCEP. - Protein crystallization buffers and precipitation agents: 1.6 M
trisodium citrate tribasic dihydrate, 2 M lithium sulfate, 3.5 M
ammonium sulfate, 100% (v/v) ethylene glycol. - Crystallization ligands: 21 mM staurosporine and
10 mM AMP. - 96–3 shallow well Intelliplates for sitting drop vapor diffusion
crystallization. - Crystallization instrument: e.g., mosquito nanoliter-scale crys-
tallization robot. - Crystal mounting: e.g., CrystalCap SPINE-style cryo-crystal
mounts. - PF-06409577 (available from Sigma [aka PZ0319]).
2.4 Enzymatic
Assays
- Kinase assay buffer: 50 mM HEPES-NaOH pH 7.4, 1 mM
EGTA, 10 mM MgCl 2 , 0.25 mM dithiothreitol (DTT), 0.01%
(v/v) Tween 20, 0.01% (w/v) bovine serum albumin (BSA).
34 Ravi G. Kurumbail et al.