期刊
PROTEOMICS
卷 15, 期 4, 页码 757-762出版社
WILEY-BLACKWELL
DOI: 10.1002/pmic.201400221
关键词
In-gel digestion; Membrane proteins; Proteomic screenings; Quantification; SWATH; Technology
资金
- Fundacao para a Ciencia e Tecnologia (FCT) [PTDC/SAU-NEU/103728/2008, PTDC/NEU-NMC/0205/2012, PEst-C/SAU/LA0001/2013-2014]
- COMPETE Programa Operacional Factores de Competitividade, QREN
- European Union (FEDER - Fundo Europeu de Desenvolvimento Regional)
- National Mass Spectrometry Network (RNEM) [REDE/1506/REM/2005]
- FCT [SFRH/BD/81495/2011, SFRH/BD/88419/2012]
- Fundação para a Ciência e a Tecnologia [SFRH/BD/88419/2012, PTDC/SAU-NEU/103728/2008] Funding Source: FCT
The quantification of large proteomes across multiple samples has become the major focus of proteomics. In addition to the advantages of in-gel digestion, the extensive time and sample handling required have precluded the use of this type of method for large quantitative screens. Therefore, an adaptation of the in-gel digestion method, termed short-GeLC, is proposed as a faster and more reproducible sample preparation method for quantitative approaches. The proposed methodology was compared with two well-established procedures for sample preparation, GeLC-MS and the classic liquid digestion followed by LC-MS, using a membrane protein-enriched sample. The results show that the short-GeLC approach substantially reduces the amount of sample handling and the overall time required for analysis compared with the gel-based methods without compromising the overall results at the protein identification level. Furthermore, the short-GeLC approach in combination with the SWATH acquisition method leads to the best quantitative results: more proteins were quantified, and the reproducibility was improved. Finally, this method performed well even on challenging samples enriched in membrane proteins.
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