Advertisement

Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis

  • Ivo ChamrádEmail author
  • Jana Uřinovská
  • Beáta Petrovská
  • Hana Jeřábková
  • René Lenobel
  • Jan Vrána
  • Jaroslav Doležel
  • Marek Šebela
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1696)

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.

Key words

Cell cycle Flow cytometry Gel electrophoresis In-gel digestion Mass spectrometry Nuclear proteome Plant nucleus Protein analysis 

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

Notes

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.

References

  1. 1.
    Meier I (2009) Functional organization of the plant cell nucleus. Springer, HeidelbergCrossRefGoogle Scholar
  2. 2.
    Erhardt M, Adamska I, Franco OL (2010) Plant nuclear proteomics – inside cell maestro. FEBS J 277:3295–3306CrossRefPubMedGoogle Scholar
  3. 3.
    Petrovska B, Sebela M, Dolezel J (2015) Inside a plant nucleus: discovering the proteins. J Exp Bot 66:1627–1640CrossRefPubMedGoogle Scholar
  4. 4.
    Petrovska B, Jerabkova H, Chamrad I et al (2014) Proteomic analysis of barley cell nuclei purified by flow sorting. Cytogenet Genome Res 143:78–86CrossRefPubMedGoogle Scholar
  5. 5.
    The International Barley Genome Sequence Consortium (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature 491:711–716Google Scholar
  6. 6.
    Thavarajah R, Mudimbaimannar VK, Elizabeth J et al (2012) Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol 16:400–405CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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 43:699–715CrossRefPubMedGoogle Scholar
  8. 8.
    Wang F, Zhu J (1990) The effect of DNA intercalators on chromatin of chicken red blood cells – differential extraction on nonhistone proteins. Cell Res 1:105–118CrossRefGoogle Scholar
  9. 9.
    Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205–214CrossRefPubMedGoogle Scholar
  10. 10.
    Kennedy-Darling J, Smith LM (2014) Measuring the formaldehyde protein-DNA cross-link reversal rate. Anal Chem 86:5678–5681CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    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 6:30CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Granvogl B, Ploscher M, Eichacker LA (2007) Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Anal Bioanal Chem 389:991–1002CrossRefPubMedGoogle Scholar
  13. 13.
    Perkins DN, Pappin DJ, Creasy DM et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567CrossRefPubMedGoogle Scholar
  14. 14.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  15. 15.
    Shevchenko A, Tomas H, Havlis J et al (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefPubMedGoogle Scholar
  16. 16.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906CrossRefPubMedGoogle Scholar
  17. 17.
    Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57CrossRefGoogle Scholar
  18. 18.
    Huang DW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13CrossRefGoogle Scholar
  19. 19.
    Tautvydas KJ (1971) Mass isolation onf pea nuclei. Plant Physiol 47:499–503CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Ivo Chamrád
    • 1
    Email author
  • Jana Uřinovská
    • 1
  • Beáta Petrovská
    • 2
  • Hana Jeřábková
    • 2
  • René Lenobel
    • 1
  • Jan Vrána
    • 2
  • Jaroslav Doležel
    • 2
  • Marek Šebela
    • 1
  1. 1.Department of Protein Biochemistry and ProteomicsCentre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký UniversityOlomoucCzech Republic
  2. 2.Centre of Plant Structural and Functional Genomics, Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental Botany AS CROlomoucCzech Republic

Personalised recommendations