UCLA

Mass spectrometry (MS)-based chemoproteomics has enabled the rapid and proteome wide discovery of many ligandable and functional amino acid residues. Many of these approaches rely on the use of a pan-reactive activity-based probe bearing an alkyne handle, such as iodoacetamide alkyne for cysteine, such that after incubation the labeled proteins can be enriched out through copper catalyzed azide-alkyne cycloaddition (CuAAC or Click) to biotin azide reagents. Over the years many labs have developed isotopically enriched biotin capture reagents such that they can use the ratio of heavy to light labeled peptide to gain insight on compound labeling of their target residue. This strategy has been highly enabling leading to the discovery of many powerful ligands targeting a variety of amino acid residues, such as cysteine. Although impactful, this strategy only allows for analysis of two samples in parallel on the mass spectrometer reducing instrument throughput. To circumvent this, isobaric tags were developed which allow for peptide level labeling of many samples in parallel, followed by combination, and injection into the instrument. At the MS1-level peptides look identical but upon MS/MS collision they fragment off a reporter ion unique to the original sample it came from. This strategy has been widely applied in the field of cysteine proteomics drastically decreasing sample acquisition time. However, because isobaric labeling occurs at the peptide level each individual sample must be taken through a unique sample preparation requiring more time and higher variability due to human error. To address these issues, we developed a click-compatible isobaric tag, which allows for protein-level sample combination, obviating the need for individual sample preparations. First, we developed a solid-phase compatible dialkoxydiphenylsilane (DADPS) reagent which we were able to slot into a traditional solid-phase peptide synthesis (SPPS) workflow to build out fully functionalized capture reagents containing: i) a biotin enrichment handle, ii) a DADPS cleavable group, iii) an azide for click capture, iv) and an amino acid handle for isotope introduction. We performed cysteine chemoproteomic analysis with these reagents and through a diagnostic ion mining pipeline identified a unique fragmentation event of the click-formed triazole to form a novel diagnostic ion. We were able to leverage this diagnostic ion as the reporter for a custom 6-plex isobaric set and utilized it for cysteine chemoproteomic analysis identifying the malononitrile hydrazone moiety as a cysteine electrophilic handle. Additionally, in this first example of our silane-based cleavable isotopically labeled proteomics (sCIP) method we identified cysteine 57 of the ADP/ATP transporter ANT2 as being targeted by the mitochondrial uncoupler FCCP which may shed light on FCCPs mechanism of action beyond its established role as a protonophore. Next, we took our work a step further and developed a single sCIP reagent that could be directly slotted into existing isobaric workflows with commercially available isobaric tags such as tandem mass tags (TMT). This method, termed sCIP-TMT, uses a sCIP reagent bearing a free n-termini, which allows for in-situ formation of sCIP-TMT conjugates that can be used directly for click conjugation. In this work we showed a direct comparison with traditional TMT-enabled cysteine chemoproteomic analysis highlighting a decrease of up to 6.5 hours of sample preparation time and >4% decrease in coefficient of variance (CV) for datasets generated using sCIP-TMT. We additionally used our method to identify and validate a ligand for the oncogene crk-like protein (CRKL) presenting a possible starting point for further drug development campaigns. Finally, we developed a 29-plex set of sCIP isobaric tags based off the previously described dimethyl leucine (DiLeu) reporter. We first planned and synthesized 29 unique dimethyl leucine analogues before incorporating these reagents into our SPPS workflow with different isotopologues of L-alanine as a balancer to generate 29 unique sCIP-DiLeu isobaric tags. Using these tags in a cysteine fragment screening workflow we identified a cysteine proximal to the active site of the serine/threonine kinase VRK1 and showed this compound actively engaged the cysteine, inhibiting kinase activity by 40% upon 20 µM treatment. Additionally, we developed a workflow to profile redox functional cysteines combining our previously described SP3-Rox approach with the well-established thermal proteome profiling (TPP) technique. Using our new method, we identified over 600 cysteines with significant thermal shifts between oxidized and reduced forms and investigated some of these target’s functions further.