Electroblotting through Enzymatic Membranes to Enhance Molecular Tissue Imaging

William T. Andrews, Adrianna N. Bickner, Fernando Tobias, Kendall A. Ryan, Merlin L. Bruening, Amanda B. Hummon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

MALDI-TOF mass spectrometry imaging (MSI) is a powerful tool for studying biomolecule localization in tissue. Protein distributions in tissue provide important histological information; however, large proteins exhibit a high limit of detection in MALDI-MS when compared to their corresponding smaller proteolytic peptides. As a result, several techniques have emerged to digest proteins into more detectable peptides for imaging. Digestion is typically accomplished through trypsin deposition on the tissue, but this technique increases the complexity of the tissue microenvironment, which can limit the number of detectable species. This proof-of-principle study explores tryptic tissue digestion during electroblotting through a trypsin-containing membrane. This approach actively extracts and enzymatically digests proteins from mouse brain tissue sections while simultaneously reducing the complexity of the tissue microenvironment (compared to trypsin deposition on the surface) to obtain an increased number of detectable peptide fragments. The method does not greatly compromise spatial location or require expensive devices to uniformly deposit trypsin on tissue. Using electrodigestion through membranes, we detected and tentatively identified several tryptic peptides that were not observed after on-Tissue digestion. Moreover, the use of pepsin rather than trypsin in digestion membranes allows extraction and digestion at low pH to detect peptides from a complementary subset of tissue proteins. Future studies will aim to further improve the method, including changing the substrate membrane to increase spatial resolution and the number of detected peptides.

Original languageEnglish (US)
Pages (from-to)1689-1699
Number of pages11
JournalJournal of the American Society for Mass Spectrometry
Volume32
Issue number7
DOIs
StatePublished - Jul 7 2021
Externally publishedYes

Keywords

  • MALDI
  • electroblot
  • electrotransfer
  • imaging
  • membranes
  • molecular scanner
  • on-Tissue digestion
  • pepsin
  • peptide extraction
  • tissue imaging
  • tryptic digestion

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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