SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.
Date
2022-08-05ICR Author
Author
Sialana, FJ
Roumeliotis, TI
Bouguenina, H
Chan Wah Hak, L
Wang, H
Caldwell, J
Collins, I
Chopra, R
Choudhary, JS
Type
Journal Article
Metadata
Show full item recordAbstract
Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.
Collections
Subject
CELMoDs
IMiDs
TMTpro
cancer cell lines
isobaric labeling
targeted protein degradation
Colorectal Neoplasms
Humans
Proteome
Proteomics
Reproducibility of Results
Workflow
Research team
Functional Proteomics
Medicinal Chemistry 4
Medicinal Chemistry 2
Prote & Metabolomics Fac
Language
eng
Date accepted
2022-07-18
License start date
2022-08-05
Citation
Journal of Proteome Research, 2022, 21 (8), pp. 1842 - 1856
Publisher
AMER CHEMICAL SOC