Isotopic labelling of peptides in tissues enhances mass spectrometric profiling

Rationale: There is a need in imaging mass spectrometry to use the acquired isotope distribution to unequivocally determine the identity of a peptide ion. A way of achieving unambiguous differentiation of ions from protonated peptides from other [M+H] ⁺ ions in a tissue would be via the direct on-tissue incorporation of ¹⁸O into peptides. Methods: Tissues were first digested with trypsin for 3h at 37°C in a humidified chamber. For the ¹⁸O-labelling of digested peptides 1μL of H ₂ ¹⁸O/50mM ammonium acetate (at pH 6.75) was added to the array of tryptic spots and incubated at room temperature for 20min. α-Cyano-4- hydroxycinnamic acid was used as a matrix modifier. The mass spectral analysis of tissue sections was carried out using a matrix-assisted laser desorption/ionisation tandem time-of-flight (MALDI-TOF-TOF) instrument. Results: On-tissue incorporation of ¹⁸O into peptides cannot be carried out during the digestion step inside a humidified chamber. After tissue digestion for 3h at 37°C in an humidified chamber, ¹⁸O labelling was carried out for 20min at room temperature (no humidified chamber). No trypsin was needed to enhance the labelling. Conclusions: For first time the feasibility of ¹⁸O-labelling of peptides in situ for tissues has been demonstrated. The method decouples protein digestion from peptide labelling and is performed in sequential steps. Furthermore, we observed that ¹⁸O incorporation produces characteristic isotopic peptide distributions, thus making facile distinguishing peptides from other tissue molecular components that ionise in the MALDI ion source.