Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics - Library Build, concentration response, structure activity relationships and in-cell treatment

ABSTRACT: In this manuscript we describe our work on the development of a label-free chemoproteomics screening platform for cysteine reactive covalent fragments on a 96 well plate format. Within this workflow, we profile cysteine reactive fragments by competition with the hyper-reactive iodoacetamide desthiobiotin (IA-DTB) in cell lysates and live cells. We employ label free quantification and data independent acquisition (DIA) on an Evosep One – Bruker timsTOF Pro. The platform was used to screen a library of 80 Cys-reactive covalent fragments in HEK293T and Jurkat cell lysate, identifying functionally relevant hit fragments for numerous cysteine sites and demonstrating the utility of the approach in expanding the ligandable proteome. We have also validated a number of selective interactions through dose response and further expanded our proteomics platform to perform hit expansion studies and in-cell validation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: George Biggs  

LAB HEAD: Jacob Bush

PROVIDER: PXD054145 | Pride | 2024-11-18

REPOSITORIES: Pride