Ltq xl mass spectrometry

DNA sequences of Tum-5 (amino acids 45–132 of tumstatin) were obtained by spliced overlap extension PCR technology. The primers used in the study were listed in Supplementary Table 1. The Tum-5 gene was inserted into the Nco I and Xho I restriction enzyme sites of pET-28a and pET-22b. Tum-5 was cloned into BamH I and Xho I restriction sites of pSmart-I (small ubiquitin-related modifier-SUMO fusion expression system) and pSmart-II (initiation factor-IF2 protein structure domain I fusion expression system). The resulting vectors were named pET-28a-Tum 5, pET-22b-Tum 5, pSmart-I-Tum 5, and pSmart-II-Tum 5. The four plasmids were transformed into E. coli BL21(DE3) and cultured at 37 °C in LB medium supplemented with 100 μg/ml ampicillin or 50 μg/ml kanamycin. Until the OD₆₀₀ value reached 0.4–0.6, IPTG was added at a final concentration of 0.3 mM to induce Tum-5 protein expression at 30 °C. Cells were harvested by centrifugation at 8000 rpm after 4 h induction. After washed twice and resuspended in a mixture of 50 mM NaH₂PO₄ and 300 mM NaCl at pH 8.0, the cells were lysed by sonication, and the supernatants and pellets were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) after centrifugation. The recombinant protein was cut with a surgical blade and identified by LTQ XL mass spectrometry (Thermo Fisher) after proteolysis.

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.