Covalent Labeling by Intact Mass Spectrometry

5 μg of purified Pin1 protein in 50 μL of 20 mM HEPES pH 7.5 and 75 mM NaCl was incubated with 5 μM of respective Pin1 inhibitors for 0–3 h. A Shimadzu XR HPLC was used to inject the entire sample onto a self-packed reverse-phase column (1/32 in outer diameter × 500 μm inner diameter, 5 cm of POROS 50R2 resin). After desalting, protein was eluted with an HPLC gradient (0%–100% B in 4 min, A = 0.2 M acetic acid in water, B = 0.2 M acetic acid in acetonitrile, flow rate = 10 μl/min) into a LTQ XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). LTQ XL MS spectra were acquired in centroid mode using the electron multipliers for ion detection. Mass spectra were deconvoluted using MagTran1.03b2 software⁴⁸.