Investigation and Development of the BODIPY-Embedded Isotopic Signature for Chemoproteomics Labeling and Targeted Profiling

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