Citation: CAO Xiaolin, GONG Jiadi, CHEN Mingxue, YU Shasha, BIAN Yingfang, CAO Zhaoyun. Analysis of rice leaves proteomes by liquid chromatography-tandem mass spectrometry based on the purification using a novel affinity detergent removal spin column[J]. Chinese Journal of Chromatography, ;2014, 32(11): 1181-1186. doi: 10.3724/SP.J.1123.2014.06035 shu

Analysis of rice leaves proteomes by liquid chromatography-tandem mass spectrometry based on the purification using a novel affinity detergent removal spin column

1.
Rice Product Quality Inspection and Supervision Center of Ministry of Agriculture, Laboratory of Quality and Safety Risk Assessment for Rice, Ministry of Agriculture, China National Rice Research Institute, Hangzhou 310006, China

Corresponding author: CAO Zhaoyun,
Received Date: 23 June 2014
Available Online: 21 July 2014
Fund Project: 中央级公益性科研院所基本科研业务专项(CNRRI2012RG007-2) (CNRRI2012RG007-2)国家粮油作物产品质量安全风险评估项目(GJFP2014006). (GJFP2014006)

A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies (>95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.
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