Integral-Omics: serial profiling of metabolome, lipidome, genome, transcriptome, whole proteome, and phosphoproteome in biopsy tissue

Integrative multi-omics studies often analyze different tissue pieces or homogenized bulk specimens, neglecting the limited size of biopsy-level specimens (

We extensively reviewed established methods for the profiling of metabolome, lipidome, genome, transcriptome, proteome, and phosphoproteome, and developed a novel integral workflow for extracting metabolites, lipids, genomic DNA, total RNA, proteins, and phosphopeptides from a biopsy-level tissue specimen (10 mg). It does not require specialized instruments. The workflow begins with lipidome and metabolome extraction, followed by gentle sonication to release RNA and proteins in a lysis buffer, while DNA remains in the precipitate. Optional phosphopeptide enrichment can be performed for phosphoproteome characterization.

From a 10 mg mouse liver sample, the Integral-Omics pipeline successfully identified 1138 lipids and 997 metabolites using MRM-based targeted mass spectrometry on a Sciex 6500+ instrument. Additionally, the pipeline yielded a total RNA extraction of 0.18% with RIN/RQN values ranging from 8.1 to 8.6, and a genomic DNA extraction of 0.14% with a main fragment size exceeding 20,000 bp. Peptides were extracted with a yield of 9.7%. Subsequently, a 30-minute DIA analysis on a Q Exactive HF instrument resulted in the identification of 48,337 peptides derived from 4652 proteins. For phosphoproteome analysis, 80 µg of peptides underwent enrichment, leading to the identification of 18,238 phosphopeptides using a 90-minute DDA LC-MS acquisition strategy on a Bruker timsTOF Pro instrument. We further applied this method to 6 colorectal adenoma and carcinoma tissue specimens (~ 10 mg each), and demonstrated its practicality in analyzing the multiomics of clinical biopsy specimens.

In summary, our approach enables the sequential profiling of metabolome, lipidome, genome, transcriptome, whole proteome, and phosphoproteome using biopsy tissue samples. This integrated methodology provides a comprehensive analysis of biomolecules, offering insights into tissue composition and biological processes.