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. Here, we describe an MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from pathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including less than 10,000 cancer cells isolated by laser-capture microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent (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 work up took less than a day. We observed a moderate trend towards lower protein identifications in long-term stored samples (>15 years) but clustering into distinct proteomic subtypes was independent of archival time. Our results underline the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and prove the feasibility of analyzing large tissue cohorts in a robust, timely and streamlined manner.

ARDD 2020: from aging mechanisms to interventions

“…In particular, single cell proteomes can be analysed to extract valuable information regarding disease mechanisms. Its integration with multiplexed imaging of human-derived tissue samples and deep learning techniques enables the creation of an advanced deep visual proteomics pipeline for discovery of novel biomarkers and effective therapeutics that could potentially be used in clinics [2]. Further, Ieva Bagdonaite, University of Copenhagen, AGING Denmark, underlined the value of investigating protein glycosylation that undergoes dynamic changes in ageassociated diseases, as well as highlighted the importance of mapping complex glycoproteome using advanced quantitative proteomics for both developing efficient therapies and biomarker discovery [3].…”

Isolating and Analyzing Protein Containing Granules from Cells

“…Here, we describe a cross‐linking approach to stabilize weak interactions in protein assemblies or condensates to allow separation or isolation from cells. Formaldehyde is cell permeable and crosslinks primary and secondary amines separated by <8 Å (Coscia et al., 2020; Tayri‐Wilk et al., 2020). Formaldehyde cross‐linking is employed to analyze protein interactomes and structure in many molecular biology protocols, including chromatin immunoprecipitation, immunocytochemistry, and mass spectrometry (Hoffman, Frey, Smith, & Auble, 2015; Li et al., 2016; Sinz, 2006; Sutherland, Toews, & Kast, 2008; Toews, Rogalski, Clark, & Kast, 2008).…”

The Implementation of Mass Spectrometry-Based Proteomics Workflows in Clinical Routines of Acute Myeloid Leukemia: Applicability and Perspectives

“…The impact of the traces of these chemicals on the mass spectrometer’s performance differs, being most detergents not compatible with downstream electrospray ionization (ESI) MS. Thus, more LC-MS/MS-friendly preparations such as those that employ formalin fixation and paraffin-embedding (FFPE) of tissue followed by cell lysis with TFE has achieved a proteomic depth of 5000–6500 proteins groups per single sample in a reproducible fashion [ 114 ]. This protocol characterized by a short sample preparation time (app.…”