Citric Acid Antigen Retrieval (CAAR) for Tryptic Peptide Imaging Directly on Archived Formalin-Fixed Paraffin-Embedded Tissue

Abstract

Imaging mass spectrometry (IMS) is a powerful technology for mapping distributions of biological molecules like proteins and peptides within tissue sections. It is therefore potentially extremely useful for the analysis of pathological conditions such as neoplastic diseases. The use of IMS is typically limited to fresh frozen tissue specimens. However, there is a high interest in the possibility of being able to analyze the tissue proteome of formalin-fixed paraffin-embedded (FFPE) specimens that have been stored together with the clinicopathological information of patients in huge archives over many decades. We have therefore developed an antigen-retrieval protocol using a high temperature citric acid buffer to allow partial reversal of FFPE protein cross-linking. Coupled with automated deposition of trypsin and matrix, our method allows the generation of meaningful peptide ion distribution images. In situ peptide fragmentation provided identification of high abundance proteins such as Actin and Collagen. Furthermore, downstream application of three different HPLC-MS strategies allowed identification of a maximum of 106 proteins, 67 of which were mass correlated to ions from IMS analysis of archived FFPE ovarian tissue. The CAAR method presented here complements previously described antigen-retrieval protocols and is an important step in being able to fully analyze the proteome of archived FFPE tissue.

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

“…[6,26] Thus, much attention is currently focused on the capacity of MALDI-IMS to provide patient-specific and/or cancerspecific information from formalin-fixed archives and, in particular, tissue micro-arrays (TMAs). [42] Dissemination of this detailed protocol, based on previously demonstrated method advancements, [29,32] will hopefully lead to the formulation of an international standard MALDI-IMS method for clinical cohorts (e.g. TMAs).…”

“…[35] 2.2 Protocol-specific solutions 2.2.1 10 mM citric acid monohydrate pH 6 [29,36] ▪ 1.05 g citric acid monohydrate (Sigma-Aldrich, Japan) ▪ Dissolve in 480 mL H 2 O ▪ pH to 6.0 using 1 M NaOH (~13 mL) (Merck, Darmstadt, Germany) ▪ Make up volume to 500 mL with H 2 O 2.2.2 100 mM ammonium bicarbonate (NH 4 [32] ▪ 0.4 pmol/mL angiotensin I [34][35][36][37][38][39][40][41][42][43] ( Figure 1. Protocol for mapping the distribution and determining the identity of tryptic peptides generated in situ.…”

“…[29,36] . 3.17 With the lid loosely placed on top, put the coplin jar in a microwave and use standard settings (i.e.…”

“…[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. [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.…”

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 .…”