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Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

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Abstract

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.

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Abbreviations

CHAPS 3:

[(3-Cholamidopropyl) dimethylamonio]-1-propanesulfonate

2-DE:

Two-dimensional gel electrophoresis

DTT:

Dithiothreitol

IEF:

Isoelectric focusing

LC:

Liquid chromatography

MS:

Mass spectrometry

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TCA:

Trichloroacetic acid

Tris:

Tris (hydroxymethyl) aminomethane

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Acknowledgments

This study has been supported grants by National Natural Science Foundation of China (31000287), by Natural Science Foundation of Higher Education of Jiangsu Province (10KJB220002) and National Natural Science Foundation of China (30930077). The authors gratefully acknowledge technical support kindly provided by the Shanghai Applied Protein Technology Co. Ltd. for their kindfuly technical support in the use of LC MS/MS.

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Authors and Affiliations

  1. Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education of the People’s Republic of China, Nanjing Forestry University, 159 Longpan Road, 210037, Nanjing, People’s Republic of China

    Yan Zhen & Jisen Shi

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  1. Yan Zhen
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  2. Jisen Shi
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Correspondence to Jisen Shi.

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Communicated by J. V. Jorrin-Novo.

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Zhen, Y., Shi, J. Evaluation of sample extraction methods for proteomic analysis of coniferous seeds. Acta Physiol Plant 33, 1623–1630 (2011). https://doi.org/10.1007/s11738-010-0697-1

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  • DOI: https://doi.org/10.1007/s11738-010-0697-1

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