An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS

Xiang Jin,Guohua Ding,Xuchu Wang,Quanliang Xie,Hongbin Li,Liping Zhu,Yanhua Tan,Chengcheng Tao,Xinyang Xu,Lili Chang

doi:10.1186/s12864-019-5658-5

Abstract

BackgroundTwo-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome.ResultsHere, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS.ConclusionOur data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Highlights

Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LCMS/Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MS)) are widely used in plant proteomics research
We found that the BPP method failed to produce high-quality 2-DE maps for cotton leaves; we used the trichloroacetic acid/acetone (TCA) method to extract total proteins for this experiment [24]
The corresponding protein yields were 1371.18 μg (0.14%) for the BPP method, 14,177.69 μg (1.42%) for the TCA method and 4698.26 μg (0.47%) for TCA combined with BPP (TCA-B) method. These results showed that the protein yield of the BPP method ranked the lowest, while the protein yield of the TCA method was the highest (Additional file 2)

Summary

Introduction

Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LCMS/MS) are widely used in plant proteomics research. These two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. Using iTRAQ, we identified 2729 differential abundant proteins in ovules from Xuzhou 142 and a fuzzless-lintless mutant (fl) cotton at the day of anthesis, indicating that very-long-chain fatty acids play an important role in cotton fibre initiation [14]. Gel-based comparative proteomic analysis of different stages of developmental initiation and somatic embryogenesis found 149 differentialy abundant proteins, indicating that stress responses might regulate the balance of reactive oxygen species in cotton through interaction with auxin during somatic embryogenesis [16]. Further study showed that the site-specific truncation of HSP70 might be involved in cotton fibre development [17]

Results

Discussion

Conclusion