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Cleavable Crosslinkers as Tissue Fixation Reagents for Proteomic Analysis.
Sara Ongay1, Miriam Langelaar-Makkinje2, Marcel P Stoop3
1Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.
Chembiochem : a European Journal of Chemical Biology
|January 23, 2018
Summary
Cleavable protein crosslinkers like DSP offer a mild alternative to formaldehyde fixation for tissue proteomics. DSP fixation improves protein extraction and identification in mass spectrometry analysis of fixed tissues.
Area of Science:
- Biochemistry
- Proteomics
- Histology
Background:
- Formaldehyde fixation is standard for tissue preservation but hinders mass spectrometry (MS) proteomic analysis due to protein crosslinking.
- Current methods for extracting proteins from formaldehyde-fixed tissues require harsh conditions, impacting sensitive modifications and yields.
Purpose of the Study:
- To evaluate cleavable protein crosslinkers as alternatives to formaldehyde for tissue fixation.
- To enable efficient protein extraction and MS proteomic analysis under mild conditions.
Main Methods:
- Investigated disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) as cleavable crosslinking fixatives.
- Assessed tissue morphology preservation and protein extraction efficiency compared to formaldehyde fixation.
- Analyzed protein recovery and identification following cleavage of crosslinkers.
Main Results:
- DST and DSP fixation preserved tissue morphology comparably to formaldehyde.
- DSP cleavage significantly improved protein recovery (18-fold) and protein identification (~20%) under mild conditions.
- DST fixation resulted in side reactions hindering protein extraction and identification.
Conclusions:
- Cleavable crosslinkers, particularly DSP, are viable alternatives to formaldehyde for tissue fixation in MS proteomics.
- DSP fixation facilitates efficient protein extraction and analysis from fixed tissues with defined modifications.
- Mild cleavage conditions preserve sensitive amino acids and post-translational modifications.
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