Fig. 3: Comprehensive proteomic profiling of membrane protein oxidation induced by BTP photocatalysis.

a Schematic illustration of the proteomic analysis workflow used to investigate the extent of oxidative modifications induced by BTP photocatalysis. The process involves a multistage search strategy for identifying O-Met and FPOP (fast photochemical oxidation of proteins) modifications. In the first search, 2,120,870 peptide-spectrum matches (PSMs) were found, and in the second search, 1,959,844 PSMs were found. Samples subjected to BTP photocatalysis and control samples were analysed and compared based on the fold change in the average precursor intensity of oxidative modifications (inset). b Proteins categorised by GO subcellular annotations into ‘membrane-specific’, located exclusively on membranes, and ‘membrane-cytosol’, found on both membranes and cytosol. The remaining cytosolic proteins were labelled ‘cytosolic’ proteins. c Volcano plot of O-Met ( + 16 Da) proteome showcasing oxidation focused on membrane-specific proteins versus cytosolic proteins. Proteins with a p-value < 0.05 and Fold Change > 2 were defined as potential oxidation targets of BTP photocatalysis. p values were calculated for Student’s one-tailed t test. d The proportions of oxidised membrane proteins across different organelles based on the O-Met proteome. ‘Others’ include plasma membranes and unidentified locations. e Overview of the 2^nd search based on FPOP modifications, showing average oxidation intensities for different amino acids. ‘All AAs’ represents the aggregated intensities of oxidative modifications of these 17 amino acids. The averaged oxidation intensities of ‘membrane-specific’, ‘membrane-cytosolic’, and ‘cytosolic’ proteins for the corresponding amino acids were presented to compare the degree of oxidation of membrane proteins and soluble proteins for each type of amino acid. The averaged oxidation intensities of three control conditions were normalised to 1. f Volcano plots of the proteome other than O-Met, contrasting membrane-specific proteins with cytosolic proteins. Stricter criteria than sole O-Met analysis (p-value < 0.01 and Fold Change > 4) were applied for robust identification of oxidised proteins. p values were calculated for Student’s one-tailed t test. Source data are provided as a Source Data file.