Objectives: The success of novel in-situ immunohistoproteomics approaches that employ Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry imaging (MSI) heavily depends on good antigen retrieval efficacy.
Methods: We have optimized the effect of various antigen retrieval buffers on peptide profiles acquired from FFPE traction alopecia scalp tissue. Heat-induced epitope retrieval (HIER) was performed using Citrate, Tris, and Tris-EDTA buffers under various conditions. MALDI-MSI analysis of the tissue was performed using a high-throughput, state-of-the-art RapiFlex MALDI TOF/TOF tissue-typer equipped with a smart beam laser.
Results: Background matrix peaks were found under most conditions except under Tris-EDTA buffer. Trypsin digestion periods longer than overnight and possibly higher concentrations are needed for Citrate and Tris. The Tris-EDTA buffer performed best for MALDI-MSI antigen retrieval at an incubation period of overnight at 60°C.
Conclusions: We have identified in this study that antigen retrieval conditions need to be optimised prior to MALDI MSI analysis of different tissue types.
Key words: Skin, MALDI MSI, antigen retrieval, buffer, mass spectrometry, FFPE