Ultraflex 3 maldi tof tof instrument

MS analysis was performed using a Bruker Ultraflex III MALDI-TOF/TOF instrument or a Bruker Microflex LRF MALDI-TOF instrument. A mixture of angiotensin II, angiotensin I, substance P, bombesin, ACTH clip 1-17, ACTH clip 18-39, and somatostatin 28 was used as peptide calibration standard. Peptide spectra were acquired in reflectron mode. The whole detection region (five pairs of parallel wells shown with brown color in Figure 1a) was manually scanned in order to capture signal from all crystals in the well area. For each detection region, 1000 laser shots at ~35 spots were probed. The detection region was identified via the crystal rim formed by the co-crystallization of MALDI matrix and sample through the camera in the MALDI TOF instrument. Since the peptide peak m/z [M+H]⁺ 1210.6 (FATVVEELFR) resulted in the largest S/N in the experiments with Bcl-2 protein solution it was chosen as the sample peptide for quantification. An isotope labeled peptide m/z [M+H]⁺ 1220.6 (FATVVEEL(d10)FR) or m/z 1217.6 (FATVVEEL(13C,15N)FR) with the same sequence was dissolved to a known concentration as the internal standard for quantification. Peak areas of the Bcl-2 digest peptide and the internal standard peptide in the MS spectra were analyzed using OriginPro software to calculate the ratio of the two and quantify Bcl-2.^{48 (link)-49 (link)}