title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics.
abstract: The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition on a TripleTOF 5600 instrument. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides.
authors.affiliation: Cambridge Systems Biology Centre and Dept. of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
authors.forename: Jakob
authors.initials: J
authors.lastname: Vowinckel
authors.affiliation: Cambridge Systems Biology Centre and Dept. of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
authors.forename: Floriana
authors.initials: F
authors.lastname: Capuano
authors.affiliation: Cambridge Systems Biology Centre and Dept. of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
authors.forename: Kate
authors.initials: K
authors.lastname: Campbell
authors.affiliation: Cambridge Systems Biology Centre and Dept. of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Deery
authors.affiliation: Cambridge Systems Biology Centre and Dept. of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
authors.forename: Kathryn S
authors.initials: KS
authors.lastname: Lilley
authors.affiliation: Cambridge Systems Biology Centre and Dept. of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK ; Division of Physiology and Metabolism, MRC National Institute for Medical Research, London, NW7 1AA, UK.
authors.forename: Markus
authors.initials: M
authors.lastname: Ralser
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