Original Article
Investigation of an optimal lysis method for the study of thymus and thymoma by mass spectrometry-based proteomics
Abstract
Background: Thymoma is the most common disease in the anterior mediastinum with malignant potentials even with low overall incidence. Although a few studies on genetic aberrations involved in thymoma have been published, a proteomic study on the landscape of the protein profiles that contributes to the systematic understanding of development of thymomas remains unseen. This is partly due to lack of generic protocols in efficiently extracting proteins from both thymus and thymoma tissues. In this paper, a one-step and highly efficient protein preparation protocol has been established and evaluated. With the use of the presented protocol, it is for the first time that over 2,900 proteins with various molecular functions and cellular locations were identified from human thymoma and thymus tissues. Application of such protocol would allow researchers to study thymus and its carcinogenesis in a comprehensive and systematic manner.
Methods: Three representative human tissue types, namely normal (thymus), Thymoma and Para-tumor were applied. Three groups of experimental procedures using different protein extraction buffers including: (I) tris buffer followed by chloroform/methanol extraction; (II) a modified RIPA buffer; (III) buffer with different concentrations of acid-labile surfactant (ALS) were compared. In total, eleven experimental settings were evaluated to optimize the protocol and the proteomes of these three tissue types were profiled using high resolution (HR) tandem mass spectrometry (MS).
Results: In this study, we found that chloroform/methanol extraction removed lipids and some lipoprotein efficiently but also resulted in a significant protein loss during the protein extraction. The modified RIPA buffer provided the highest protein yield in the extraction step but after an additional acetone precipitation step required for MS analysis, the total protein yield was significantly compromised and therefore was not considered for further applications. Using the buffers with various ALS concentrations, proteins were extracted in a reliable manner, which showed a positive correlation between the amounts of extracted proteins and the applied ALS concentrations. Proteomics analysis of all the three tissues lysed by ALS led to the identification of 2,902 proteins in total, of which about 1,271 (43.8%) proteins were identified only in thymoma.
Conclusions: We present here a simple protein extraction protocol, specifically designed for proteomics analysis for human thymus and thymoma tissues. Application of this method in together with an HR-MS analysis using Orbitrap Fusion, has led to identification of 2,000+ proteins from the thymoma tissue and 1,000+ proteins from the thymus tissue. Comprehensive proteomic profiling of such a large number of proteins with various molecular functions and cellular locations could significantly broaden and deepen our understanding of the mechanisms about thymus carcinogenesis, metastasis, and possible recurrence.
Methods: Three representative human tissue types, namely normal (thymus), Thymoma and Para-tumor were applied. Three groups of experimental procedures using different protein extraction buffers including: (I) tris buffer followed by chloroform/methanol extraction; (II) a modified RIPA buffer; (III) buffer with different concentrations of acid-labile surfactant (ALS) were compared. In total, eleven experimental settings were evaluated to optimize the protocol and the proteomes of these three tissue types were profiled using high resolution (HR) tandem mass spectrometry (MS).
Results: In this study, we found that chloroform/methanol extraction removed lipids and some lipoprotein efficiently but also resulted in a significant protein loss during the protein extraction. The modified RIPA buffer provided the highest protein yield in the extraction step but after an additional acetone precipitation step required for MS analysis, the total protein yield was significantly compromised and therefore was not considered for further applications. Using the buffers with various ALS concentrations, proteins were extracted in a reliable manner, which showed a positive correlation between the amounts of extracted proteins and the applied ALS concentrations. Proteomics analysis of all the three tissues lysed by ALS led to the identification of 2,902 proteins in total, of which about 1,271 (43.8%) proteins were identified only in thymoma.
Conclusions: We present here a simple protein extraction protocol, specifically designed for proteomics analysis for human thymus and thymoma tissues. Application of this method in together with an HR-MS analysis using Orbitrap Fusion, has led to identification of 2,000+ proteins from the thymoma tissue and 1,000+ proteins from the thymus tissue. Comprehensive proteomic profiling of such a large number of proteins with various molecular functions and cellular locations could significantly broaden and deepen our understanding of the mechanisms about thymus carcinogenesis, metastasis, and possible recurrence.
