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

Abstract

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.

Analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue Via Proteomic Techniques and Misconceptions of Antigen Retrieval

“…In the case of FFPE tissue, a tissue section can be analyzed via IMS to determine where individual proteins or peptides are on a pixel-by-pixel basis. Standard IMS workflows involve coating a slide-mounted tissue section that has been either deparaffinized and digested with trypsin (18) or simply washed (19) before addition of an organic acid matrix and recrystallizing the matrix and analyte together. The matrix allows for absorption of UV energy from the laser, thus generating ions that are measured by their time of flight.…”

Mass Spectrometry Imaging of Biomolecular Information

“…FFPE samples can be analyzed and imaged after direct tryptic digestion 154 or after heat-induced antigen retrieval, dewaxing an digestion 155 . MALDI MSI of FFPE tissue has been reported in great numbers recently 10,[156][157][158][159][160][161][162][163][164][165][166][167][168][169][170][171][172][173] . To mention just one example: ovarian cancer FFPE samples were investigated to describe progression, metastases and other factors of cancer development by topographic parameters and molecular information 169 .…”

MALDI mass spectrometric imaging meets “omics”: recent advances in the fruitful marriage

“…106 Hence, more and more researchers are employing various on-tissue digestions techniques, developed for the specific tissue type they are working on 25,73,107,108 Additionally in 2013, a multi-center study has been set up to improve and validate the current protocols under the roof of the COST action BM1104. 106 Hence, more and more researchers are employing various on-tissue digestions techniques, developed for the specific tissue type they are working on 25,73,107,108 Additionally in 2013, a multi-center study has been set up to improve and validate the current protocols under the roof of the COST action BM1104.…”

“…4 Due to the increasing applications of ultra violet (UV) MALDI MSI in various "omics" field, the information, which matrix is suitable for which family of compound classes and mass range can be easily found in the literature. enzymatic digestion, 25,26 derivatization, 27 transfer to functionalized surfaces), 28 and instrumentation for the application of the matrix. [5][6][7] Hence, Table 1 provides a snapshot of the current state-of-art of common and new UV MALDI MSI matrices, subdivided into the corresponding "omics" fields.…”