Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Sep 4

PMID 37665466

Authors

Frank Rolfs

Richard R de Goeij-de Haas

Jaco C Knol

Sander R Piersma

Connie R Jimenez

Affiliations

Soon will be listed here.

Abstract

Proteogenomic analysis is emerging as an advantageous tool to assist personalized therapy decisions in clinical health care and integrates complementary information from the genome, transcriptome, and (phospho)proteome. A prerequisite for such analysis is a workflow for the simultaneous isolation of DNA, RNA, and protein from a single sample that does not compromise the different biological molecules and their examination. Focusing on the phosphoproteomic aspect of this workflow, we here provide detailed information on our protocol, which is based on commonly used acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction with RNA-Bee. We describe the necessary steps for biopsy collection, cryoprocessing, and protein extraction. We further share our practice on protein digestion and cleanup of small samples (200 μg protein) and describe settings for automated IMAC-based phosphopeptide enrichment with the AssayMAP Bravo platform.

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