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Isotopic labelling of peptides in tissues enhances mass spectrometric profiling
Author(s) -
Santos Hugo M.,
Kouvonen Petri,
Capelo Jose L.,
Corthals Garry L.
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5325
Subject(s) - chemistry , labelling , peptide , trypsin , mass spectrometry , chromatography , desorption , digestion (alchemy) , matrix assisted laser desorption/ionization , tandem mass spectrometry , isotopic labeling , biochemistry , enzyme , organic chemistry , adsorption
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 18 O into peptides. METHODS Tissues were first digested with trypsin for 3 h at 37 °C in a humidified chamber. For the 18 O‐labelling of digested peptides 1 μL of H 2 18 O/50 mM ammonium acetate (at pH 6.75) was added to the array of tryptic spots and incubated at room temperature for 20 min. 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 18 O into peptides cannot be carried out during the digestion step inside a humidified chamber. After tissue digestion for 3 h at 37 °C in an humidified chamber, 18 O labelling was carried out for 20 min at room temperature (no humidified chamber). No trypsin was needed to enhance the labelling. CONCLUSIONS For first time the feasibility of 18 O‐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 18 O incorporation produces characteristic isotopic peptide distributions, thus making facile distinguishing peptides from other tissue molecular components that ionise in the MALDI ion source. Copyright © 2012 John Wiley & Sons, Ltd.