Crops ›› 2016, Vol. 32 ›› Issue (2): 50-56.doi: 10.16035/j.issn.1001-7283.2016.02.009

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Comparison of Preparation and Digestion for Crop Leaf Proteomes

Su Xiaoqin1,3,Wei Hongru2,3,Duan Shuangmei1,Zhao Ming1,Pan Yinghong3   

  1. 1 College of Longrun Puer Tea,Yunnan Agricultural University,Kunming 650201,Yunnan,China
    2 College of Agronomy,Hebei Agriculture University,Baoding 071000,Hebei,China
    3 Institute of Crop Science,Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement,Beijing 100081,China
  • Received:2015-10-27 Revised:2016-03-04 Online:2016-04-15 Published:2018-08-26
  • Contact: Ming Zhao

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Abstract:

The sample preparation, extraction and protein digestion, are key steps in proteomics analysis. In order to improve the efficiency and repeat ability of crop leaf proteome samples, the leaves of rice, wheat, soybean and corn were respectively extracted with six buffers (pH4.5 acetic acid salt, pH7.0 phosphate, pH8.0 Tris-HCl, pH8.5 Tris- HCl phenol, pH9.0 carbonate, and urea-thiourea), and pretreatment with TCA-acetone. And then, the proteins extracted from wheat leaves and bovine serum albumin were digested by standard trypsin digestion and microwave-assisted digestion. SDS-PAGE and protein extraction rates showed that TCA-acetone precipitation combined with urea-thiourea extraction was suitable for preparation of rice and wheat leaf protein, and urea-thiourea extraction without TCAacetone precipitation was suitable for preparation of corn and soybean leaf protein. LC-MS-MS analysis of digested peptides suggested that standard and microwave-assisted digestion were both effective in proteomics analysis. Together, the protocol for preparation of proteome samples from leaves of rice, wheat, soybeans and corn was developed. This study confirmed that, the method by using the appropriate protein extraction method combining microwave-assisted digestion, could effectively improve the coverage and efficiency of crop leaf proteome analysis.

Key words: Proteome, Sample preparation, Protein digestion, Crop, Leaf

Fig.1

SDS-PAGE of crop proteins extracted with different buffers"

Fig.2

Yields of proteins extracted with different buffers and pretreatment methods"

Fig.3

SDS-PAGE of crop proteins extractedwith urea-thiourea"

Fig.4

SDS-PAGE of crop proteins extracted with urea-thiourea"

Tab.1

BSA digested with standard and microwave-assisted digestion methods"

酶切方法Digestion 酶切时间(h)
Digestion time		 牛血清白蛋白覆盖率（%）BSA coverage
0漏切位点
0 missed cleavages		 1漏切位点
1 missed cleavages		 2漏切位点
2 missed cleavages
传统酶切Standard digestion 20.0 74.55 21.82 3.64
微波辅助酶切Microwave-assisted digestion 3.3 70.77 23.82 6.15

Fig.5

Total ion chromatogram (TIC) and mass spectrometry results of wheat proteins digested with standard and microwave-assisted methods"

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