Development of a novel encyclopaedic peptide spectral library using the liquid fraction of sheep blood

Abstract

Proteome measurements derived from tandem mass spectrometry (MS/MS) are a powerful means to detect and quantify known and novel proteins. This methods-based thesis has developed a novel peptide spectral library (PSL) that can be applied to detect multiple proteins within complex MS/MS datasets, including protein markers of inflammation in ovine plasma. Such biomarkers permit better definition of the mammalian response to disease.The potential use of the PSL is in targeted proteomics applications such as sequential window acquisition of all theoretical fragment ion mass spectra (SWATH)-MS analysis and other targeted proteomics techniques requiring a spectral library for quantitation of proteins found in sheep plasma or serum. A major advantage of SWATH-MS analysis of proteins is that the current alternative, the enzyme-linked immunosorbent assays (ELISA), can only measure a single protein for each kit, has to be validated for each species and is costly and cumbersome.In light of the field’s current status, this thesis sought to address four key objectives: (1) develop a comprehensive method to characterise the ovine circulating acellular proteome; (2) investigate various protein fractionation techniques to enhance protein identification yields from plasma and serum samples; (3) optimise a bioinformatics workflow for constructing a PSL as a tool for identifying proteins and for future proteogenomics uses, and; (4) apply the PSL to samples obtained from healthy and ill sheep to identify candidate markers of inflammation.To achieve the above objectives, a baseline PSL was established using serum samples from healthy sheep. This PSL was broadened using a range of protein fractionation techniques, including acetone precipitation, partial organic precipitation with acetonitrile (ACN), combinatorial peptide ligand library enrichment (ProteoMiner™, Bio-Rad), as well as off-gel isoelectric focussing. Sample fractions were processed to tryptic peptides and analysed using nano-liquid chromatography electrospray ionisation tandem mass spectrometry (nanoLC-ESI-MS/MS) on an Eksigent nanoLC coupled to a quadrupole time-of-flight (QqTOF) mass spectrometer (TripleTOF 5600+, SCIEX) operated in a data-dependent acquisition (DDA) mode. Data from diseased and archived endotoxin-treated experimental sheep samples, as well as in silico predicted and synthesised peptides of five proinflammatory cytokines, namely Interleukin 6 (IL-6), Interleukin 3 (IL-3), Interleukin 1a (IL-1α), Interleukin 1b (IL-1β) and tumour necrosis factor-alpha (TNF-α) were later processed and added to create the encyclopaedic PSL.For all DDA experiments, MS/MS data were searched against an Ovis aries UniProtKB (Universal Protein Resource Consortium Knowledgebase) database using ProteinPilot™ Software (SCIEX) primarily, and then secondarily using Mascot (Matrix Science) search engine to identify proteins. These protein identifications (IDs) were validated using PeptideShaker (CompOmics, Inc) proteomics informatics software.Following establishment of the PSL, plasma samples from sheep, before and after endotoxin-treatment, were analysed by data independent acquisition (DIA), namely SWATH-MS. Data were analysed using the SWATH™ MicroApp 2.0 (SCIEX) alongside the PSL to profile proteins in plasma samples in the two cohorts.The primary ProteinPilot™ search identified 41,288 distinct peptides from 3,195,890 spectra at a false discovery rate (FDR) threshold of 1%. Together with secondary analysis in Mascot and validation in PeptideShaker, these spectra enabled the identification of 398 proteins in the nascent PSL. Using PeptideShaker identification results from DDA experiments, the baseline PSL and the acetone precipitation experiments yielded 133 and 102 protein IDs, respectively. The ACN precipitation workflow resulted in 198 protein IDs. The ProteoMiner™ workflow yielded 305 protein IDs, representing 56.4% increase in protein IDs, compared to undepleted samples. The off-gel experiment yielded 70 protein IDs, 15 (21.4%) of which were attributed to fractionation compared to 55 protein IDs from crude serum. The diseased and endotoxin-treated sheep experiments yielded 183 and 84 protein IDs, respectively, and collectively contributed 80 protein IDs not detected in healthy sheep samples. Validated spectrum annotation matches were obtained from two peptides each of IL-6, IL-3, IL-1α, IL-1β and TNF-α in the PSL from the in silico predicted and synthesised proinflammatory cytokines experiment.The use of SWATH analysis enabled the quantitation of 243 proteins in sheep plasma and also revealed that the samples of the sheep model were non-identical between individuals and yet they were expected to be alike. Forty well-recognised acute phase proteins (APP) were quantitated and 42 other proteins were potentially endotoxin-induced candidate markers of inflammation.This research has developed the first PSL resource designed for measuring the proteinaceous portion of blood from healthy and diseased sheep. Through its application, the first use of SWATH-MS is reported herein to identify candidate protein markers of inflammation in sheep’s plasma. Through this work, the feasibility of simultaneously identifying many protein alterations in a cost-effective manner and with widely available tools has been reinforced, with potential applications in veterinary pathology, animal welfare and in screening laboratory animals before inclusion in experimental groups to minimise differences. In future, the scope of the PSL can be broadened by including proteomic and genomic data from cellular components of blood and other tissues to complement ovine genome annotation efforts.