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General Session 3: Biological oceanography & global change |
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Marine metaproteomics: Comparative evaluation of protein extraction protocols
GS3-21-S
Lingfen Kong* , State Key Laboratory of Marine Environmental Science/College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
Yingbao Gai, State Key Laboratory of Marine Environmental Science/College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
Xue Cheng, State Key Laboratory of Marine Environmental Science/College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
Dazhi Wang, State Key Laboratory of Marine Environmental Science/College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
Presenter Email: konglingfen513@gmail.com
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| Culture-independent techniques such as LC-MS/MS-based metaproteomic analyses are utilized increasingly for the studies on microbial compositions and functions of complex environmental samples. Although several protein extraction approaches have been compared by conducting on soil, activated sludge and tissue samples, few reports are concerning about seawater samples, especially samples filtered through GF/F glass filters. Due to the complexity of seawater samples, the exhaustive extraction of proteins is a major challenge, and it is meaningful, therefore, for marine metaproteomic researches to investigate different extraction methods in terms of improving extraction efficiencies and sources of bias. Here, we compared marine protein extraction protocols in terms of their extraction efficiency for GF/F glass filters and PES (polyether sulfone). First, three preprocessing methods were compared, including (a) sonioation method, (b) liquid nitrogen grounding and subsequent sonication method, (c) crushed by MP FastPrep. For GF/F glass filters, crushing samples by MP FastPrep were the most efficiency preprocessing method, while for PES the best choice was treating samples by liquid nitrogen grounding and subsequent sonication. Based on the most suitable preprocessing methods, five different protein extraction procedures were applied based on (a) Trizol extraction, (b) Lysis buffer extraction, (c) SDS-phenol extraction, (d) NoviPure kit extraction, and (e) B-PER extraction. Proteins were analyzed by LC-MS/MS and the number of unique spectra as well as the number of assigned proteins for each protocol was compared. For both membrane materials, isolation manners with NoviPure kit extraction resulted in a relative high number of proteins. Our study revealed that a critical evaluation of extraction protocol is crucial for the quality of metaproteomics data, especially in highly complex samples like seawater samples. |
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