title: Short GeLC-SWATH: a fast and reliable quantitative approach for proteomic screenings.
abstract: 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.
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Faculty of Sciences and Technology, University of Coimbra, Portugal.
authors.forename: Sandra Isabel
authors.initials: SI
authors.lastname: Anjo
authors.forename: Cátia
authors.initials: C
authors.lastname: Santa
authors.forename: Bruno
authors.initials: B
authors.lastname: Manadas
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D020543:Proteome
keywords: In-gel digestion; Membrane proteins; Proteomic screenings; Quantification; SWATH; Technology