Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry

Abstract

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…Afterward, several teams proved that it was possible to perform the AR directly on tissue sections. These applications were mainly dedicated to MALDI imaging analyses (Bonnel et al, 2011;Casadonte and Caprioli, 2011;Gustafsson et al, 2010). However, more recently, Longuespée used citric acid antigen retrieval (CAAR) before shotgun proteomics associated to global profiling proteomics .…”

Matrix‐assisted laser desorption/ionization imaging protocol for in situ characterization of tryptic peptide identity and distribution in formalin‐fixed tissue

“…[14] ▪ Mount the section in one smooth motion to prevent section curling or folding. ▪ The paraffin edge of the section will stick to the slide.…”

“…[6,29] The methods necessary to prepare FFPE samples, perform MALDI-IMS, analyze data and characterize the resulting peptide features have not yet been standardized and are largely developed in-house by individual laboratories. [6,7,14,30,31] As such, the need to add to the limited number of detailed protocols in the field [14] prompted the current manuscript, in which the methods developed and published in two previous manuscripts by our group [29,32] have been described in a single detailed experimental workflow combining tryptic peptide MALDI-IMS and subsequent liquid chromatographic/tandem mass spectrometric (LC/MS/MS) identification of the peptides which localize to a region of interest (e.g. tumour tissue).…”

“…[26] In order to circumvent the formalin-induced cross-links most groups have either adopted a proteolytic enzyme [7,28] or antigen retrieval and enzyme combination treatment. [6,14] Recently, applied methods demonstrating the usage of antigen retrieval in combination with tryptic proteolytic digestion have shown marked success in reproducibly generating peptides which are discriminatory for known histological features. [6,29] The methods necessary to prepare FFPE samples, perform MALDI-IMS, analyze data and characterize the resulting peptide features have not yet been standardized and are largely developed in-house by individual laboratories.…”

“…[1,[10][11][12] The advantages of the MALDI-IMS approach include the ability to analyze hundreds of analytes in a single experiment without prior knowledge of tissue composition, or the use of antibodies. [13,14] Furthermore, MALDI-IMS can utilize as little as one tissue section per analysis, making it ideal for precious samples. [8,15] To date, the most clinically relevant application of MALDI-IMS has been the acquisition of in situ molecular profiles which can be used to classify tissue types and thereby provide patient specific information.…”