title: Comparing Simplification Strategies for the Skeletal Muscle Proteome.
abstract: Skeletal muscle is a complex tissue that is dominated by the presence of a few abundant proteins. This wide dynamic range can mask the presence of lower abundance proteins, which can be a confounding factor in large-scale proteomic experiments. In this study, we have investigated a number of pre-fractionation methods, at both the protein and peptide level, for the characterization of the skeletal muscle proteome. The analyses revealed that the use of OFFGEL isoelectric focusing yielded the largest number of protein identifications (>750) compared to alternative gel-based and protein equalization strategies. Further, OFFGEL led to a substantial enrichment of a different sub-population of the proteome. Filter-aided sample preparation (FASP), coupled to peptide-level OFFGEL provided more confidence in the results due to a substantial increase in the number of peptides assigned to each protein. The findings presented here support the use of a multiplexed approach to proteome characterization of skeletal muscle, which has a recognized imbalance in the dynamic range of its protein complement.
authors.affiliation: Division of Health Research, University of the Highlands and Islands, Inverness IV2 3JH, UK. beth.geary@uhi.ac.uk.
authors.forename: Bethany
authors.initials: B
authors.lastname: Geary
authors.affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK. isyoung@liverpool.ac.uk.
authors.forename: Iain S
authors.initials: IS
authors.lastname: Young
authors.affiliation: Division of Applied Medicine, University of Aberdeen, Aberdeen AB25 2ZD, UK. p.cash@abdn.ac.uk.
authors.forename: Phillip
authors.initials: P
authors.lastname: Cash
authors.affiliation: Division of Health Research, University of the Highlands and Islands, Inverness IV2 3JH, UK. phil.whitfield@uhi.ac.uk.
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Whitfield
authors.affiliation: Division of Health Research, University of the Highlands and Islands, Inverness IV2 3JH, UK. mary.doherty@uhi.ac.uk.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Doherty
keywords: FASP; OFFGEL; ProteoMiner; protein equalization; proteome simplification; skeletal muscle