Abstract
In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.
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Abbreviations
- ACN:
-
Acetonitrile
- AmBic:
-
Ammonium bicarbonate
- APS:
-
Ammonium persulfate
- CBB:
-
Coomassie Brilliant Blue
- CHCA:
-
α-Cyano-4-hydroxycinnamic acid
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DNase:
-
Deoxyribonuclease
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediaminetetraacetic acid
- ESI:
-
Electrospray ionization
- FA:
-
Formic acid
- FSC:
-
Forward-scattered light
- GO:
-
Gene ontology
- HPLC:
-
High-performance liquid chromatography
- LC-ESI-MS:
-
Liquid chromatography coupled to electrospray ionization mass spectrometry
- LC-MALDI-MS:
-
Liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry
- LC-MS/MS:
-
Liquid chromatography coupled to tandem mass spectrometry
- LSB:
-
Loading sample buffer
- MALDI:
-
Matrix-assisted laser desorption/ionization
- MS:
-
Mass spectrometry
- MS/MS:
-
Tandem mass spectrometry
- PMSF:
-
Phenylmethanesulfonylfluoride
- SDS:
-
Sodium dodecyl sulfate
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- StageTip:
-
Stop-and-go Tip
- TFA:
-
Trifluoroacetic acid
- UHR-Q-TOF:
-
Ultra-high-resolution quadrupole time-of-flight mass spectrometer
References
Meier I (2009) Functional organization of the plant cell nucleus. Springer, Heidelberg
Erhardt M, Adamska I, Franco OL (2010) Plant nuclear proteomics – inside cell maestro. FEBS J 277:3295–3306
Petrovska B, Sebela M, Dolezel J (2015) Inside a plant nucleus: discovering the proteins. J Exp Bot 66:1627–1640
Petrovska B, Jerabkova H, Chamrad I et al (2014) Proteomic analysis of barley cell nuclei purified by flow sorting. Cytogenet Genome Res 143:78–86
The International Barley Genome Sequence Consortium (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature 491:711–716
Thavarajah R, Mudimbaimannar VK, Elizabeth J et al (2012) Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol 16:400–405
Sutherland BW, Toews J, Kast J (2008) Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. J Mass Spectrom 43:699–715
Wang F, Zhu J (1990) The effect of DNA intercalators on chromatin of chicken red blood cells – differential extraction on nonhistone proteins. Cell Res 1:105–118
Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205–214
Kennedy-Darling J, Smith LM (2014) Measuring the formaldehyde protein-DNA cross-link reversal rate. Anal Chem 86:5678–5681
Ngoka LC (2008) Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci 6:30
Granvogl B, Ploscher M, Eichacker LA (2007) Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Anal Bioanal Chem 389:991–1002
Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Shevchenko A, Tomas H, Havlis J et al (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57
Huang DW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13
Tautvydas KJ (1971) Mass isolation onf pea nuclei. Plant Physiol 47:499–503
Acknowledgment
This work was supported by a grant from the National Program of Sustainability I (LO1204) from the Ministry of Education, Youth and Sports of the Czech Republic.
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Chamrád, I. et al. (2018). Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis. In: Mock, HP., Matros, A., Witzel, K. (eds) Plant Membrane Proteomics. Methods in Molecular Biology, vol 1696. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7411-5_4
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DOI: https://doi.org/10.1007/978-1-4939-7411-5_4
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7409-2
Online ISBN: 978-1-4939-7411-5
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