Maldi tof ms

Recombinant human FKBP12 was obtained as described in Supplementary Methods. Purified FKBP12 (5 μM) was incubated with reagent (10 μM) in the absence or presence of rapamycin (20 μM) in HEPES buffer (50 mM, pH 7.2) at 37 °C. Aliquots at different time points were taken and then desalted using a Ziptip-C4 (Merck), and the labelling yields were determined by MALDI-TOF MS (matrix: CHCA).

Wild-type and R273H p53 core domains were de-salted against 20 mM ammonium acetate buffer by using 10 K concentration columns (Vivaspin, GE Healthacare, Chicago, IL). Twenty µM of the purified protein were incubated with 0 µM (control), 50, 100 or 200 µM MQ for 15 min at 21 °C. R175H core domains were de-salted by ZipTip C4 resin tips for MALDI-ToF MS (Merck Millipore, Billerica, MA) following the manufacturer’s protocol. 3.2 µM of R175H protein were treated with 0 µM (control), 10, 25 or 50 µM of MQ for 15 min at 21 °C. 5% formic acid (1:1 volume ratio) was added to the samples to increase the ionization sensitivity. Samples were analyzed by LTQ XL mass spectrometry (Thermo Fisher Scientific, Waltham, MA) fitted with an automated nanospray source (TriVersa Nanomate, Advion Biosciences, Ithaca, NY) using nanoelectrospray chips with spraying nozzels. The ion source was controlled using the Chipsoft 8.3.1 software (Advion Biosciences, Ithaca NY). Three microliters of each sample were loaded into a 96-well plate and injection volume was one and a half microliters. Full scan spectra were collected at the m/z 500–2000 in positive ion mode. The mass spectra of each sample were acquired in profile mode over 4 min. The spectra were analyzed using XCalibur^TM Software (Thermo Fisher Scientific, Waltham, MA). Deconvoluted ESI spectra are presented.

A weak cation exchange magnetic bead (WCX MB) kit was used (Bioyong Tech, Beijing, China). Alpha-cyano-4-hydroxycinnamic acid (CHCA) was dissolved in 100% ethanol (chromatographic grade) and 100% acetone (chromatographic grade) to freshly prepare the sample matrix for MALDI-TOF MS (Bruker Bio-sciences, Bremen, Germany). The suspension in the WCX kit was mixed with samples by shaking. The peptides were separated from the magnetic beads by eluting and beating, and the eluted peptide samples were transferred to a 0.5-mL sample tube (Bioyong Tech). Subsequently, 5 mL of CHCA substrate solution (0.4 g/L, dissolved in acetone and ethanol) and 0.8–1.2 μL of elution peptides were mixed, and 0.8–1.2 μL of this mixture was applied to a metal target plate and dried at room temperature (Bioyong Tech). Finally, the prepared samples were analyzed by MALDI-TOF MS (Bioyong Tech). We used a three-peptide mixture (monoisotopic molecular weights of 1533.8582, 2465.1989, and 5730.6087 Da, Product Numbers P2613, A8346, and I6279, respectively; Sigma-Aldrich, USA) to calibrate the MALDI-TOF MS prior to analyzing our samples. Profile spectra were acquired of 400 shots of laser per sample. A range of 1000–10000 Da peptide molecular weight was detected. Each sample was analyzed thrice, and the mean value of each sample was used for analysis.