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Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics - 80 compound screen (HEK293T and Jurkat)

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): timsTOF Pro 2

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: George Biggs

LAB HEAD: Jacob Bush

PROVIDER: PXD054127 | Pride | 2024-11-18

REPOSITORIES: Pride

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			Action	DRS
	DC2461_ELB00032_20220318_DIA_H00AGKH_P01_A02_S1-A2_1_2090.d.rar	Other
	DC2461_ELB00032_20220318_DIA_H00AGKH_P01_A03_S1-A3_1_2029.d.rar	Other
	DC2461_ELB00032_20220318_DIA_H00AGKH_P01_A04_S1-A4_1_1994.d.rar	Other
	DC2461_ELB00032_20220318_DIA_H00AGKH_P01_A05_S1-A5_1_1963.d.rar	Other
	DC2461_ELB00032_20220318_DIA_H00AGKH_P01_A06_S1-A6_1_2042.d.rar	Other

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