Cleavable crosslinkers as tissue fixation reagents for proteomic analysisOngay, S., Langelaar-Makkinje, M., Stoop, M. P., Liu, N., Overkleeft, H., Luider, T. M., Groothuis, G. M. M. & Bischoff, R., 11-Apr-2018, In : ChemBioChem. 19, p. 736-743 8 p.
Research output: Contribution to journal › Article › Academic › peer-review
Formaldehyde fixation is widely used for long-term storage of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering mass spectrometry (MS)-proteomic analyses. In the present work we evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation as well as efficient protein extraction for MS-proteomics under mild conditions. We investigated disuccinimidyl tartrate (DST) and dithiobis[succinimidylpropionate] (DSP) as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups leading to amide bond formation and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP). Our results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to formaldehyde.Cleavage of DSP improves protein recovery from fixated tissue by a factor of 18 and increases the number of identified proteins by approximately 20% under mild extraction conditions compared to formaldehyde-fixated paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, while effective, resulted in side reactions that prevented effective protein extraction and interfered with protein identification.
|Number of pages||8|
|Early online date||22-Jan-2018|
|Publication status||Published - 11-Apr-2018|
- Journal Article