Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies

Frank Rolfs; Richard R. de Goeij-de Haas; Jaco C. Knol; Sander R. Piersma; Connie R. Jimenez

doi:https://doi.org/10.1007/978-1-0716-3457-8_16

Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies

Frank Rolfs, Richard R. de Goeij-de Haas, Jaco C. Knol, Sander R. Piersma, Connie R. Jimenez

Research output: Contribution to journal › Article › Academic › peer-review

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.

Original language	English
Pages (from-to)	285-302
Number of pages	18
Journal	Methods in molecular biology (Clifton, N.J.)
Volume	2718
DOIs	https://doi.org/10.1007/978-1-0716-3457-8_16
Publication status	Published - 2023

Keywords

AGPC
Biopsy
Mass spectrometry
Multi-omics
Phosphoproteomics
RNA-Bee
Single sample
Tissue

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https://doi.org/10.1007/978-1-0716-3457-8_16

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title = "Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies",

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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AU - Rolfs, Frank

AU - de Goeij-de Haas, Richard R.

AU - Knol, Jaco C.

AU - Piersma, Sander R.

AU - Jimenez, Connie R.

N1 - Publisher Copyright: © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023.

PY - 2023

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N2 - 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.

AB - 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.

KW - AGPC

KW - Biopsy

KW - Mass spectrometry

KW - Multi-omics

KW - Phosphoproteomics

KW - RNA-Bee

KW - Single sample

KW - Tissue

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ER -

Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies

Abstract

Keywords

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