Investigation and\nDevelopment of the BODIPY-Embedded\nIsotopic Signature for Chemoproteomics Labeling and Targeted Profiling

A common\ngoal in mass spectrometry-based chemoproteomics is to\ndirectly measure the site of conjugation between the target protein\nand the small molecule ligand. However, these experiments are inherently\nchallenging due to the low abundance of labeled proteins and the difficulty\nin identifying modification sites using standard proteomics software.\nReporter tags that either generate signature fragment ions or isotopically\nencode target peptides can be used for the preemptive discovery of\nlabeled peptides even in the absence of identification. We investigated\nthe potential of BODIPY FL azide as a click chemistry enabled chemoproteomics\nreagent due to the presence of boron and the unique 1:4 natural abundance\nratio of <sup>10</sup>B:<sup>11</sup>B. The isotopes of boron encode\nBODIPY-labeled peptides with a predictable pattern between the monoisotopic\n(M) and M+1 peaks. BODIPY-labeled peptides were identified in MS1\nspectra using an R script that filters for the signature <sup>10</sup>B:<sup>11</sup>B intensity ratio and mass defect. Application of\nthe boron detection script resulted in three times the labeled peptide\ncoverage achieved for a BODIPY-conjugated BSA sample compared with\nuntargeted data-dependent acquisition sequencing. Furthermore, we\nused the inherent HF neutral loss signature from BODIPY to assist\nwith BODIPY-modified peptide identification. Finally, we demonstrate\nthe application of this approach using the BODIPY-conjugated BSA sample\nspiked into a complex <i>E. coli</i>. digest. In summary,\nour results show that the commercially available BODIPY FL azide clicked\nto alkyne-labeled peptides provides a unique isotopic signature for\npinpointing the site(s) of modification with the added potential for\non- or off-line UV or fluorescence detection.