Fig. 4: Validation of representative arginylation sites.
From: An unbiased proteomic platform for ATE1-based arginylation profiling
a, Synthetic peptide validation of seven arginylation sites. b, Representative MS2 spectra indicating RM12 arginylation on Cys45 with tri-oxidation (C45 RO3) and its MS1 summary (upper, center and lower: 75, 50 and 25 percentiles, respectively). Its first monoisotopic peak in MS1 scans (n = 44 technical scans) from a representative run is set at 1 for normalization. Relative intensities of other isotopic peaks (n = 43, 38, 30, 18, 2, 44, 44, 40, 30, 9 and 0 technical scans) are displayed. c, Open E24 in ERO1A and E17 in SSBP are arginylated in an in-bacteria arginylation system. Protein and ATE1 are coexpressed in E. coli for in-bacteria arginylation. The protein was purified and digested for proteomics analysis. d, Arginylation dependency of CALR, ERO1A and SSBP sites on endogenous ATE1 (n = 1 biological replicate). Anti-ATE1 was used to detect the expression of endogenous ATE1. β-tubulin was used as a loading control. Anti-Flag antibody was used to detect expressions of CALR, ERO1A and SSBP proteins. EICs of arginylated peptide in each protein in WT and KO cells after pull-down and proteomics are provided. e, Relative arginylation levels of CALR, ERO1A and SSBP sites after cooverexpression of ATE1. Protein was purified by antibody pull-down experiment followed by proteomics analysis. Peak areas of arginylated peptides were normalized to the sample with the highest signal and relative ratios are displayed. Different amounts (0, 0.5 and 1 µg) of ATE1 plasmids were used for transfection. β-tubulin was used as a loading control. Anti-Flag antibody was used to detect expressions of CALR, ERO1A and SSBP proteins. KO, ATE1 KO; Std, standard peptide.
