Abstract
BackgroundUnlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum.MethodsSerum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chromatography nano electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF® 5600+, SCIEX). Proteins were identified using ProteinPilot™ (SCIEX) and Mascot (Matrix Science) software based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1% software by searching a subset of the Universal Protein Resource Knowledgebase (UniProtKB) database (http://www.uniprot.org). PeptideShaker (CompOmics, VIB-UGent) searches were used to validate protein identifications from ProteinPilot™ and Mascot.ResultsProteinPilot™ and Mascot identified 245 and 379 protein groups (IDs), respectively, and PeptideShaker validated 133 protein IDs from the entire dataset. Since Mascot software is considered the industry standard and identified the most proteins, these were analysed using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification tool revealing the association of 349 genes with 127 protein pathway hits. These data are available via ProteomeXchange with identifier PXD004989.ConclusionsThese results demonstrated for the first time the feasibility of characterising the ovine circulating acellular proteome using nanoLC-nanoESI-MS/MS. This peptide spectral data contributes to a protein library that can be used to identify a wide range of proteins in ovine serum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-017-0119-z) contains supplementary material, which is available to authorized users.
Highlights
Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states
In 2005, liquid chromatography tandem mass chromatography (LC-tandem mass spectrometry (MS/MS)) data from multiple sample preparation techniques and protein sequence search engines for the Human Plasma Proteome Project (HPPP) from 18 laboratories worldwide collectively identified 3,020 plasma proteins based on a minimum of 2-high-scoring peptides [19, 20]
Protein IDs were obtained using ProteinPilotTM [55] to search a UniProtKB composite database of Ovis aries, Bos taurus and Capra hircus with a results quality of false discovery rate (FDR) ≤1%; ≥ 2 peptides for a protein to be considered confidently identified as the highest scoring member of the protein group
Summary
There is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. In 2005, liquid chromatography tandem mass chromatography (LC-MS/MS) data from multiple sample preparation techniques and protein sequence search engines for the Human Plasma Proteome Project (HPPP) from 18 laboratories worldwide collectively identified 3,020 plasma proteins based on a minimum of 2-high-scoring peptides [19, 20]. This number of protein IDs from HPPP studies was subsequently revised to 889 [19, 21]. A similar approach to that used in the preceding study was considered attractive to be used in exploring the circulating acellular proteome of sheep
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