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.
Keywords: AGPC; Biopsy; Mass spectrometry; Multi-omics; Phosphoproteomics; RNA-Bee; Single sample; Tissue.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.