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

Abstract

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 E. coli. 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.

Figure 1

(A) Chemical structure of BODIPY clicked to a theoretical 500 Da probe coupled to a tryptic peptide, and simulated doubly charged isotopic distributions of theoretical unlabeled (C₉₉H₁₅₅N₂₇O₃₀S) and BODIPY-labeled (C₁₄₃H₂₁₆BF₂N₃₇O₃₆S) peptides with 20 averagine amino acids. (B) Percent relative intensity of the M to M+1 peak for simulated BODIPY- versus unlabeled peptides (Note: M/M+1 (%) data that exceeds 100% not shown for unlabeled peptides <20 amino acids). (C) Exact mass difference between the M and M+1 peak for BODIPY- versus unlabeled peptides.

Figure 2

(A) MS1 spectrum of representative BODIPY peptide C*ASIQK demonstrates significant HF neutral loss. (B) The effect of decreasing % RF on BODIPY HF neutral loss and absolute intact peptide signal in MS1 spectra.

Figure 3

MS2 spectrum of BODIPY-labeled peptide EAC*FAVEGPK at HCD 30 shows HF and 2HF neutral loss and retention of the ¹⁰B peak in fragment ions.

Figure 4

(A) Workflow summary for the computational detection of boron encoded peptides (B) BODIPY peptide coverage achieved with MS1 and data dependent acquisition (DDA) searched with the boron script vs SEQUEST.

Figure 5

MS2 spectrum of BODIPY-labeled peptide TC*VADESHAGCEK identified using targeted profiling.