Quantitative mass spectrometry approaches have been a valuable tool in neuroproteomics, being an important ally for the deeper understanding of proteome modulation of the nervous system. Although there are several quantitative mass spectrometry approaches, all of them still require the digestion of the proteins into peptides making this step critical for the success of this type of analysis. By turning into quantitative, these methods are not only focused on the capacity to improve the depth of proteome coverage but most importantly making it in a reproducible way. In line with several improvements in digestion procedures, the short GeLC approach was presented which consists in an adaptation of the common in-gel digestion methods, in which the electrophoretic separation is performed in approximately 1–2 cm of the gel. Therefore, short GeLC retains most of the advantages of in-gel digestion, namely, its high efficiency and compatibility, in a very reproducible method that proves to be particularly advantageous for quantitative mass spectrometry analyses. Moreover, the short GeLC approach combined with SWATH acquisition has been revealed as a promising method for reliable quantitative screenings in particular when applied to challenging samples such as membrane-enriched samples and to samples of limited amount such as biofluids.
In this chapter, a detailed description of the short GeLC-SWATH pipeline is presented and complemented with the presentation of some of its different applications in the neuroproteomics field. Among different applications, some examples were selected that can demonstrate the vast versatility of the short GeLC-SWATH, namely, its application in (1) the differential proteome analysis of brain tissues and biofluids, (2) the study of the interactome of plasma membrane receptors, and (3) its application in the evaluation of receptors’ cleavage by proteases. With these examples, the use of short GeLC-SWATH with difficult samples is covered, including membrane protein-enriched samples and samples with a large dynamic range or enriched in particular proteins, and its application in very complex experimental designs.
Short GeLC SWATH-MS In-gel digestion Quantitative neuroproteomics Mass spectrometry
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This work was supported by Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-NMC/112183/2009, PTDC/NEU-NMC/0205/2012, PTDC/NEU-SCC/7051/2014, UID/NEU/04539/2013, UID/BIM/04773/2013, POCI-01-0145-FEDER-007440) and co-financed by “COMPETE Programa Operacional Factores de Competitividade,” QREN, the European Union (FEDER—Fundo Europeu de Desenvolvimento Regional) and The National Mass Spectrometry Network (RNEM) (REDE/1506/REM/2005). Sandra I. Anjo and Cátia Santa are supported by FCT Ph.D. fellowships (SFRH/BD/81495/2011 and SFRH/BD/88419/2012).
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