In Situ Imaging of Tryptic Peptides by MALDI Imaging Mass Spectrometry Using Fresh-Frozen or Formalin-Fixed, Paraffin-Embedded Tissue

Abstract

Tryptic peptide imaging is a primary workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and has led to new information reporting highly multiplexed protein localization. Technological advances within the last few years have produced robust tools for automated spraying of both matrix and enzymes. When combined with high-mass-resolution and high-mass-accuracy instrumentation, studies now generally result in two-dimensional mapping of well over 1,000 peptide peaks. This protocol describes sample preparation, spraying, and application of enzymes and matrices, and MALDI FT-ICR instrumental considerations for two-dimensional mapping of tryptic peptides from fresh-frozen or formalin-fixed, paraffin-embedded tissue sections. Procedures for extraction of tryptic peptides from tissue sections for LC-MS/MS identification are also described. © 2018 by John Wiley & Sons, Inc.

Keywords: MALDI imaging mass spectrometry; formalin-fixed; imaging mass spectrometry; paraffin-embedded tissue imaging; peptide identification for imaging mass spectrometry; proteomics; tissue imaging; tryptic peptide imaging.

Figure 1

Example of humidity chamber setup in culture dish. A) Paper towel (Wypall X60) is placed on the bottom of the dish, cut to fit. Two Kimwipes are rolled into bundles and placed either end of the dish. Distilled water is used to saturate the paper towel and both Kimwipes. B) Example of condensation that should be seen on the cell culture dish prior to placing samples into the humidity chamber to digest.

Figure 2

Experimental workflow for generating image data by on-tissue tryptic digestion.

Figure 3

Correlation of MALDI IMS data to LC-MS/MS data by high mass accuracy. MALDI IMS uses calibration of the instrument before each imaging run, combined with the lockmass function to stabilize calibration during imaging. LC-MS/MS uses daily calibration to maintain high resolution, high accurate mass (HR/AM). Data is also recalibrated post acquisition to ensure the highest mass accuracy. MALDI IMS and LC-MS/MS are matched by precursor mass accuracy within ± 1 ppm. Protein identification is made using sequence information obtained by LC-MS/MS or by MALDI MS/MS.

Figure 4

Extraction of peptides from an entire tissue slice for identification by LC-MS/MS proteomics. Solvent is placed on the tissue forming a bead and allowed to incubate for 3 min, covered. A pipette tip is inserted at a 45° angle on the edge of the tissue and bead of solvent. The solvent is aspirated into the pipette tip and placed in a microcentrifuge tube.

Figure 5

Extraction of peptides from a target area on the tissue for identification by LC-MS/MS proteomics. A) A small amount of solvent (200–500 nl) is placed by pipette on the target region, forming a bead, shown here on mouse heart. B) The solvent is ejected and withdrawn from the target region 5–10 times for extraction without moving the pipette tip from a position perpendicular to the tissue. The solvent is then placed in a low protein binding microcentrifuge tube until LC-MS/MS.

Figure 6

Example images of expected output shown as MALDI IMS of tryptic peptides from mouse heart. A) H&E stain of heart. B) Example images of tryptic peptide expression showing 110 images out of 1,469 monoisotopic peaks (excluding isotopes 2 and 3 of isotopic envelopes). C) Example single images. Mouse heart tissue section donated by Dr. Christine Kern, Medical University of South Carolina. Mouse heart was fixed in 4% paraformaldehyde, paraffin embedded, and sectioned at 5 µm thickness.