Evaluation of a Pooling Chemoproteomics Strategy with an FDA-Approved Drug Library

Abstract

Chemoproteomics is a key platform for characterizing the mode of action for compounds, especially for targeted protein degraders such as proteolysis targeting chimeras (PRO-TACs) and molecular glues. With deep proteome coverage, multiplexed tandem mass tag-mass spectrometry (TMT-MS) can tackle up to 18 samples in a single experiment. Here, we present a pooling strategy for further enhancing the throughput and apply the strategy to an FDA-approved drug library (95 best-in-class compounds). The TMT-MS-based pooling strategy was evaluated in the following steps. First, we demonstrated the capability of TMT-MS by analyzing more than 15 000 unique proteins (> 12 000 gene products) in HEK293 cells treated with five PROTACs (two BRD/BET degraders and three degraders for FAK, ALK, and BTK kinases). We then introduced a rationalized pooling strategy to separate structurally similar compounds in different pools and identified the proteomic response to 14 pools from the drug library. Finally, we validated the proteomic response from one pool by reprofiling the cells via treatment with individual drugs with sufficient replicates. Interestingly, numerous proteins were found to change upon drug treatment, including AMD1, ODC1, PRKX, PRKY, EXO1, AEN, and LRRC58 with 7-hydroxystaurosporine; C6orf64, HMGCR, and RRM2 with Sorafenib; SYS1 and ALAS1 with Venetoclax; and ATF3, CLK1, and CLK4 with Palbocilib. Thus, pooling chemoproteomics screening provides an efficient method for dissecting the molecular targets of compound libraries.

Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics

“…3 B and 2D ). As a 36% NCE is widely reported as optimal for MS2-based TMT experiments 58 , 87 , 88 , these findings support that the sCIP functionality does not substantially change the behavior of the piperidine reporter ion. Interestingly, the cysteine desulfurization ion is predominant at lower NCEs (Fig.…”

Application of PROTACs in target identification and validation

“…To avoid off-target effects, some researchers employed mass spectrometry-based proteomic techniques to study the degradation specificity of PROTACs. TMT-labeled quantitative proteomic analysis is the most often used method and has been successfully applied to specifically targeted degradation studies involving K-RAS [92], SGK-3 [93], LRRK2 [94], SMARCA2 [70], BRD/BET, FAK, ALK, and BTK [95]. This method has also driven the development of molecular glue discovery platforms [96].…”