Abstract
Main conclusion
Ammonium sulfate is well known to salt out proteins at high concentrations. The study revealed that it can serve to increase by 60% the total number of identified carbonylated proteins by LC–MS/MS.
Abstract
Protein carbonylation is a significant post-translational modification associated with reactive oxygen species signaling in animal and plant cells. However, the detection of carbonylated proteins involved in signaling is still challenging, as they only represent a small subset of the proteome in the absence of stress. In this study, we investigated the hypothesis that a prefractionation step with ammonium sulphate will improve the detection of the carbonylated proteins in a plant extract. For this, we extracted total protein from the Arabidopsis thaliana leaves and subjected the extract to stepwise precipitation with ammonium sulfate to 40%, 60%, and 80% saturation. The protein fractions were then analyzed by liquid chromatography–tandem mass spectrometry for protein identification. We found that all the proteins identified in the non-fractionated samples were also found in the prefractionated samples, indicating no loss was incurred during the prefractionation. About 45% more proteins were identified in the fractionated samples compared to the non-fractionated total crude extract. When the prefractionation steps were combined with the enrichment of carbonylated proteins labeled with a fluorescent hydrazide probe, several carbonylated proteins, which were unseen in the non-fractionated samples, became visible in the prefractionated samples. Consistently, the prefractionation method allowed to identify 63% more carbonylated proteins by mass spectrometry compared to the number of carbonylated proteins identified from the total crude extract without prefractionation. These results indicated that the ammonium sulfate-based proteome prefractionation can be used to improve proteome coverage and identification of carbonylated proteins from a complex proteome sample.
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Data availability
The data underlying this article are available in the article and in its online supplementary material. The data can also be found here: MassIVE MSV000088920, MSV000090559, ProteomeXchange PXD031898, PXD037611.
Abbreviations
- AMS:
-
Ammonium sulfate
- CPLL:
-
Combinatorial hexapeptide ligand libraries
- Cy5.5-hydrazide:
-
Cyanine 5.5 hydrazide
- Cy7.5-hydrazide:
-
Cyanine 7.5 hydrazide
- 1D-PAGE:
-
One-dimensional polyacrylamide gel electrophoresis
- GO:
-
Gene ontology
- LC–MS/MS:
-
Liquid chromatography mass spectrometry
- MS:
-
Mass spectrometry
- PEG:
-
Polyethylene glycol
- PTM:
-
Post-translational modification
- ROS:
-
Reactive oxygen species
- SDS-PAGE:
-
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
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Acknowledgements
The authors wish to express their gratitude to Sylvie Bourassa and Victor Fourcassié for the LC-MS/MS data collection at Proteomics Platform of the CHU de Québec Research Center (Quebec, QC, Canada). The authors also thank Souaibou Yaouba for helping draw the chemical reactions.
Funding
This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant Program, grant number DGECR-2019-00304 to TDM.
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425_2023_4083_MOESM1_ESM.tif
Supplementary file1 Fig. S1: Gel pictures of the two additional biological replicates of the total crude protein from Arabidopsis leaves and the fractionated samples. (TIF 1365 KB)
425_2023_4083_MOESM2_ESM.tif
Supplementary file2 Fig. S2: Gel pictures of the carbonylated proteins in total crude protein from Arabidopsis leaves and the AMS-fraction samples. Total protein crude extracts were derivatized with Cy5.5-hydrazide and protein samples of the AMS-fractions (40%, 60% and 80%) were derivatized with Cy7.5-hydrazide. Then, 10 micrograms of total protein from crude extract derivatized with Cy5.5-hydrazide (lanes 1, 4 and 7), 40%-AMS fraction (lane 2), 60%-AMS fraction (lane 5), and 80%-AMS fraction (lane 8) derivatized with Cy7.5-hydrazide were separated within a 12.5% SDS-PAGE. For comparison between the samples, two samples were mixed and loaded in a few lanes containing 5 µg of each. Hence, there were crude extract derivatized with Cy5.5-hydrazide + 40%-AMS fraction derivatized with Cy7.5-hydrazide (lane 3); 5 µg crude extract derivatized with Cy5.5-hydrazide + 5 µg of 60%-AMS fraction derivatized with Cy7.5-hydrazide (lane 6); 5 µg crude extract derivatized with Cy5.5-hydrazide + 5 µg of 80%-AMS fraction derivatized with Cy7.5-hydrazide (lane 9). (TIF 3185 KB)
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Tola, A.J., Missihoun, T.D. Ammonium sulfate-based prefractionation improved proteome coverage and detection of carbonylated proteins in Arabidopsis thaliana leaf extract. Planta 257, 62 (2023). https://doi.org/10.1007/s00425-023-04083-6
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DOI: https://doi.org/10.1007/s00425-023-04083-6