Figure 4. Cardiac fibroblasts convert ubiquitin(1–76) to ubiquitin(1–74).

SHR cardiac fibroblasts were treated with ubiquitin(1–76) (U-76; 1000 nmol/L) for 24 hours, and the medium was collected and processed for analysis by mass spectrometry. Samples were loaded onto a polar reverse phase column connected to a Shimadzu HPLC/LCMS-2010 system. Separation was conducted with a linear gradient from 20% acetonitrile to 60% acetonitrile in the presence of 0.1% formic acid at flow rate 0.4 ml/min. The mass spectrometer was operated in the positive mode with selected ion monitoring. The chromatographic peak (total ion current) corresponding to the retention time of ubiquitin(1–76) and ubiquitin(1–74) (7.46 to 9.00 minutes) was scanned for multiple charged ions (M¹²⁺ to M⁷⁺): for ubiquitin(1–76), 714.80, 779.80, 857.80, 952.80, 1071.80, and 1224.80 m/z, respectively; for ubiquitin(1–74), 705.30, 769.50, 846.20, 940.20, 1057.50, 1208.50 m/z, respectively). The m/z versus signal intensity plot of the selected ions unequivocally identifies ubiquitin(1–74) as a product of ubiquitin(1–76).