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

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the means to map the in situ distribution of tryptic peptides in formalin-fixed clinical tissue samples. The ability to analyze clinical samples is of great importance to further developments in the imaging field. However, there is a requirement in this field of research for additional methods describing the characterization of tryptic peptides by MALDI imaging. METHODS AND RESULTS: This protocol gives highly detailed instructions, with examples, for (1) successfully performing tryptic peptide MALDI imaging on formalin-fixed cancer tissue using a MALDI-TOF/TOF MS instrument, (2) tentatively generating identifications through nLC/MS/MS, and (3) validating these identifications by in situ MS/MS of peptides of interest. CONCLUSIONS: This protocol provides a detailed and straightforward description of the methods required for groups new to MALDI imaging to begin analysis of formalin-fixed clinical samples.