Top-down characterization of genetically encoded Bcl-XL phosphorylation and phosphomimetics using electron capture dissociation

Genetically encoded phosphoserine and non-hydrolyzable phosphonate-containing phosphorylation mimics play a crucial role in studying phosphorylation-dependent processes. Genetic code expansion (GCE) allows the creation of site-specifically modified proteins with non-hydrolyzable and native phosphoserine. The precise engineering of phosphorylated proteins enables the measurement of phosphorylation-specific effects on protein function. Combining GCE with top-down mass spectrometry, enables an in-depth characterization of phospho-proteoforms which informs downstream binding studies. Here, we employ top-down analysis to characterize Bcl-XL, a protein central to cellular apoptosis. Within minutes of data collection, we performed sequence and impurity analysis on Bcl-XL variants containing phosphoserine and phospho mimics. These results underscore the importance of top-down characterization to inform studies on phosphorylation-dependent processes.

Top-down analysis of Bcl-xL was performed using an Agilent 6545XT quadrupole time-of-flight (QTOF) mass spectrometer equipped with an ExD cell for electron fragmentation. Bcl-XL variants were expressed and purified by the GCE4All Center using amber codon reassignment to create pure, site-specifically modified Bcl-XL. Intact masses were determined using Mass Hunter BioConfirm. ExDViewer v4.5.11 was used for top-down sequence analysis and localization of modifications. Downstream binding studies were accomplished using isothermal titration calorimetry (ITC).

In contrast to traditional bottom-up techniques that demand extensive sample preparation, our approach minimizes these requirements and reveals residue-level differences among samples within a 1-minute data collection period. We isolated the 19+ precursor of intact Bcl-XL proteins (~23kDa) with encoded phosphoserine, non-hydrolyzable phosphoserine, serine, glutamine, or alanine in position 62. Utilizing electron capture dissociation (ECD), we achieved 85-90% sequence coverage for all Bcl-XL variants including site-specific evidence of mutations and modifications. The spectrum included both ECD and collision-induced dissociation (CID) fragmentation ions which contributed to overall sequence coverage. For the wild-type Bcl-xL sample, the fragmentation results yielded 84% coverage considering ECD ions alone and 87% coverage when considering both ECD and CID-type ions. In addition, we show that the 2 Da difference between non-hydrolyzable phosphoserine with a phosphonate group and genuine phosphoserine can be differentiated using top-down electron capture mass spectrometry. The confirmation of modification identity & site via top-down ECD revealed a contaminant in one phosphomimetic sample, leading to re-analysis of downstream binding studies using ITC. Effective analysis of unknown contaminants and precise mapping of modifications demonstrate the utility of pairing genetic code expansion with top-down analysis for phosphoprotein characterization.