A streamlined mass spectrometry–based proteomics workflow for large‐scale FFPE tissue analysis

Abstract

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner.

Sarcoma Molecular Pathological Diagnosis of FFPE Tissues without Dewaxing based on Lipidomic Profiles using SpiderMass Technology

“…Alongside, strategies were developed to perform proteomics by AR combined with enzymatic digestion (most often trypsin) from FFPE (37,38). Protocols have been optimized over the years and nowadays thousands of proteins can be identi ed from archived tissues (39). In 2007, strategies were also proposed to unlock mass spectrometry imaging (MSI) of proteins from FFPE tissues (21) and nowadays protocols for protein analysis from FFPE tissue are highly reproducible (23).…”

Quantitative proteomics of coeliac gut during 14-day gluten challenge: low-level baseline inflammation despite clinical and histological normality predicts subsequent response

“…The field of MS-based analysis of clinical FFPE material is developing rapidly and currently receiving much attention in particular in the field of cancer diagnostics. [18] While the applicability for diagnosis may yet be limited, this approach represent a highly valuable complementary tool to classical tissue histology analysis.…”

“…[14][15][16][17] Advances in mass spectrometry (MS)-based analysis of proteins extracted from FFPE biopsies is paving way for the use of archival clinical material collected for pathology assessment to perform tissue proteome analysis. [18] We recently demonstrated that proteome analysis of FFPE tissue sections from untreated and treated coeliac disease patients accurately captures disease hallmarks and processes that we know change upon disease remission. [19] By use of laser-capture microdissection (LCM), specific regions or compartments can be isolated from FFPE tissue sections for proteome analysis.…”

The Role of Diagnostic Biomarkers, Omics Strategies, and Single-Cell Sequencing for Nonalcoholic Fatty Liver Disease in Severely Obese Patients

“…While actual single-cell proteomics of human livers have not been done yet, recent technological advances has opened up for large-scale proteomics in formalin-fixed, paraffinembedded liver biopsy tissue, with great promise for further understanding of hepatic cells' functional contributions to liver biology [82].…”