VPBrowse: Genome-based representation of MS/MS spectra to quantify 10,000 bovine proteins.

SWATH is a data acquisition strategy acclaimed for generating quantitatively accurate and consistent measurements of proteins across multiple samples. Its utility for proteomics studies in nonlaboratory animals, however, is currently compromised by the lack of sufficiently comprehensive and reliable public libraries, either experimental or predicted, and relevant platforms that support their sharing and utilization in an intuitive manner. Here we describe the development of the Veterinary Proteome Browser, VPBrowse (http://browser.proteo.cloud/), an on-line platform for genome-based representation of the Bos taurus proteome, which is equipped with an interactive database and tools for searching, visualization, and building quantitative mass spectrometry assays. In its current version (VPBrowse 1.0), it contains high-quality fragmentation spectra acquired on QToF instrument for over 36,000 proteotypic peptides, the experimental evidence for over 10,000 proteins. Data can be downloaded in different formats to enable analysis using popular software packages for SWATH data processing whilst normalization to iRT scale ensures compatibility with diverse chromatography systems. When applied to published blood plasma dataset from the biomarker discovery study, the resource supported label-free quantification of additional proteins not reported by the authors previously including PSMA4, a tissue leakage protein and a promising candidate biomarker of animal's response to dehorning-related injury.

Non-CG DNA methylation marks the transition from pupa to adult in Helicoverpa armigera.

DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of 'housekeeping' genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows.

The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin; thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal; n = 30) and Aged (Primiparous; n = 20) dairy cows (Bos taurus) of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV; p value = 3.302e-07) and 0.95 (plasma; p value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (*p = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (***p = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle.

Automated Proteomics Workflows for High-Throughput Library Generation and Biomarker Detection Using Data-Independent Acquisition.

Sequential window acquisition of all theoretical mass spectra-mass spectrometry underpinned by advanced bioinformatics offers a framework for comprehensive analysis of proteomes and the discovery of robust biomarkers. However, the lack of a generic sample preparation platform to tackle the heterogeneity of material collected from different sources may be a limiting factor to the broad application of this technique. We have developed universal and fully automated workflows using a robotic sample preparation platform, which enabled in-depth and reproducible proteome coverage and characterization of bovine and ovine specimens representing healthy animals and a model of myocardial infarction. High correlation (R2 = 0.85) between sheep proteomics and transcriptomics datasets validated the developments. The findings suggest that automated workflows can be employed for various clinical applications across different animal species and animal models of health and disease.

Prophylactic and therapeutic vaccination protects sperm health from Chlamydia muridarum-induced abnormalities.

Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFß were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, ß-casein and ß-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

Exosomal Cargo May Hold the Key to Improving Reproductive Outcomes in Dairy Cows.

The reproductive status of dairy cows remains a challenge for dairy farmers worldwide, with impaired fertility linked to a significant reduction in herd profitability, due in part to impaired immunity, increased metabolic pressure, and longer postpartum anestrous interval (PPAI). Exosomes are nanovesicles released from a variety of cell types and end up in circulation, and carry proteins, bioactive peptides, lipids, and nucleic acids specific to the place of origin. As such, their role in health and disease has been investigated in humans and animals. This review discusses research into exosomes in the context of reproduction in dairy herds and introduces recent advances in mass-spectrometry (MS) based proteomics that have a potential to advance quantitative profiling of exosomal protein cargo in a search for early biomarkers of cattle fertility.

Effects of increased scrotal temperature on semen quality and seminal plasma proteins in Brahman bulls.

Environmental temperature has effects on sperm quality with differences in susceptibility between cattle subspecies and breeds, but very little is known about the seminal plasma protein (SPP) changes resulting from testicular heat stress. Scrotal insulation (SI) for 48 hr was applied to Brahman (Bos indicus) bulls. Semen was collected at 3-day intervals from before, until 74 days post-SI. The changes in sperm morphology and motility following SI were comparable to previously reported and differences were detected in measures of sperm chromatin conformation as early as 8 days post-SI. New proteins spots, in the SPP two-dimensional (2-D) gels, were apparent when comparing pre-SI with 74 days post-SI, and SPP identified as associated with mechanisms of cellular repair and protection. Similar trends between 2-D gel and Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) data was observed, with SWATH-MS able to quantify individual SPP that otherwise were not resolved on 2-D gel. The SPP assessment at peak sperm damage (21-24 days) showed a significant difference in 29 SPP (adjusted p < .05), and identified six proteins with change in abundance in the SI group. In conclusion both spermatozoa and SPP composition of bulls are susceptible to temperature change incurred by SI, and SPP markers for testicular heat insults may be detected.

Structural elucidation of hydroxy fatty acids by photodissociation mass spectrometry with photolabile derivatives.

RATIONALE: Eicosanoids are short-lived bio-responsive lipids produced locally from oxidation of polyunsaturated fatty acids (FAs) via a cascade of enzymatic or free radical reactions. Alterations in the composition and concentration of eicosanoids are indicative of inflammation responses and there is strong interest in developing analytical methods for the sensitive and selective detection of these lipids in biological mixtures. Most eicosanoids are hydroxy FAs (HFAs), which present a particular analytical challenge due to the presence of regioisomers arising from differing locations of hydroxylation and unsaturation within their structures. METHODS: In this study, the recently developed derivatization reagent 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+ ) was applied to a representative set of HFAs including bioactive eicosanoids. Photodissociation (PD) mass spectra obtained at 266 nm of 4-I-AMPP+ -modified HFAs exhibit abundant product ions arising from photolysis of the aryl-iodide bond within the derivative with subsequent migration of the radical to the hydroxyl group promoting fragmentation of the FA chain and facilitating structural assignment. RESULTS: Representative polyunsaturated HFAs (from the hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid families) were derivatized with 4-I-AMPP+ and subjected to a reversed-phase liquid chromatography workflow that afforded chromatographic resolution of isomers in conjunction with structurally diagnostic PD mass spectra. CONCLUSIONS: PD of these complex HFAs was found to be sensitive to the locations of hydroxyl groups and carbon-carbon double bonds, which are structural properties strongly associated with the biosynthetic origins of these lipid mediators.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Characterisation of the circulating acellular proteome of healthy sheep using LC-MS/MS-based proteomics analysis of serum.

BACKGROUND: 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. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum. METHODS: Serum 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. RESULTS: ProteinPilot™ 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. CONCLUSIONS: These 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.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule.

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4µ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Identification and characterisation of moderately thermostable diisobutyl phthalate degrading esterase from a Great Artesian Basin Bacillus velezensis NP05.

Phthalate esters are known to be endocrine disrupting chemicals and are documented to pollute environments. Enzymatic degradation of PAEs is a potential bioremedial strategy to manage contamination. Thermostable bioremedial enzymes have advantages in enzyme manufacturing and storage. In this study, we identified, overexpressed, and characterised a moderately thermostable para-nitrobenzyl esterase from whole genome sequencing of a Bacillus velezensis NP05 from the Great Artesian Basin, capable of sequential 2-step hydrolysis of diisobutyl phthalate. The pnbA enzyme has a molecular weight of 55.14 kDa and pI of 5.31. It preferentially degrades para-nitrophenyl butanoate and has an optimal pH of 7-8. The pnbA esterase has an optimal temperature of 55 °C with a half-life of 4 h. Using HPLC we found that pnbA (0.122 U) can hydrolyse 0.83 mM of DIBP within 25 min. Lastly, pnbA is potentially a more economically viable candidate for enzymatic bioremediation of diisobutyl phthalate as a free enzyme.

Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition.

SCOPE: Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. METHODS AND RESULTS: This study develops a pipeline using advanced proteomics and transcriptomics to enable cross-species comparison of milk and IF EVs. The number of nanoparticles per mL of IF is significantly reduced compared to unprocessed CM. 130 proteins and 514 miRNAs are differentially abundant between HM and CM EVs. While 90% of CM EV miRNAs are also identified in IF EVs, only 20% of CM EV proteins are identified in IF EVs. CONCLUSIONS: This workflow identifies key species-specific differences that can be used to optimize IF recipes and enhance infant nutrition. Improved preservation of EV functional molecular cargo in IF products is of critical importance to retaining molecular drivers of good health and should be the focus of future investigations.

Addressing accuracy and precision issues in iTRAQ quantitation.

iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.

Investigation of cattle plasma proteome in response to pain and inflammation using next generation proteomics technique, SWATH-MS.

Pain assessment in farm animals has primarily relied on a combination of behavioral and physiological responses, although these are relatively subjective and difficult to quantify. It is essential to develop more effective biomarkers of pain in production animals since they are frequently exposed to routine surgical husbandry procedures. More effective biomarkers of pain would improve welfare, limit the loss of productivity associated with pain and permit better assessment of analgesics. This study aimed to investigate the use of a modern mass spectrometry data independent acquisition strategy, termed Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS), to detect candidate protein biomarkers that are known to associate with nociceptive and inflammatory processes in cattle, which could then be used to assess the efficacy of potential analgesics. Calves were randomly divided into two groups that were either surgically dehorned or subjected to restraint stress, without provision of anaesthesia or analgesia in accordance with current industry standards. Samples were analysed before and after dehorning at multiple timepoints. Significant changes in protein concentrations were detected predominantly at 24 and 96 h following dehorning, including kininogens, proteins associated with the coagulation and complement cascades and serine protease inhibitors. Gene ontology analysis revealed that the identified candidate biomarkers were associated with stress, wound healing, immune response, blood coagulation and the inflammatory and acute phase responses, which could be expected following surgical damage to tissues, but can now be more objectively assessed. These results offer more definitive and quantitative monitoring of response to tissue injury induced pain and inflammation.

Plasma proteomic changes in response to surgical trauma and a novel transdermal analgesic treatment in dogs.

Assessment of pain responses and inflammation during animal surgery is difficult because traditional methods, such as visual analogue scores, are not applicable while under anaesthesia. Acute phase proteins (APPs), such as C-reactive protein and haptoglobin, that are typically monitored in veterinary research, do not show a significant change until at least 2 h post-surgery and therefore, immediate pathophysiological changes are uncertain. The current study used sequential window acquisition of all theoretical mass spectra (SWATH-MS) to investigate plasma proteome changes that occur immediately following surgery in dogs and also to assess the efficacy of a novel transdermal ketoprofen (TK) formulation. Castration was chosen as surgical model in this study. The procedure was performed on twelve dogs (n = 6 in two groups) and blood samples were collected at 0 h, 1 and 2 h after surgery for proteomic analysis. Following surgery, there was a general downregulation of proteins, including complement C- 3, complement factor B, complement factor D, transthyretin, and proteins associated with lipid, cholesterol, and glucose metabolisms, reflecting the systemic response to surgical trauma. Many of these changes were diminished in the transdermal group (TD) since ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), inhibits prostanoids and the associated chemotactic neutrophil migration to site of tissue injury. SIGNIFICANCE: SWATH-MS Proteomic analysis revealed significant changes in plasma proteins, predominantly involved in early acute phase and inflammatory response at 1 & 2 h after surgery in castrated dogs. Pre-operative application of transdermal ketoprofen formulation had reduced the systemic immune response, which was confirmed by negligible alteration of proteins in transdermal treated group. A key outcome of this experiment was studying the efficacy of a novel transdermal NSAID formulation in dogs.

Data-Independent Acquisition Enables Robust Quantification of 400 Proteins in Non-Depleted Canine Plasma.

Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry has allowed for quantitative profiling of a significant number of plasma proteins in humans and several animal species. Enabling SWATH in dogs enhances human biomedical research as a model species, and significantly improves diagnostic and disease monitoring capability. In this study, a comprehensive peptide spectral library specific to canine plasma proteome was developed and evaluated using SWATH for protein quantification in non-depleted dog plasma. Specifically, plasma samples were subjected to various orthogonal fractionation and digestion techniques, and peptide fragmentation data corresponding to over 420 proteins was collected. Subsequently, a SWATH-based assay was introduced that leveraged the developed resource and that enabled reproducible quantification of 400 proteins in non-depleted plasma samples corresponding to various disease conditions. The ability to profile the abundance of such a significant number of plasma proteins using a single method in dogs has the potential to accelerate biomarker discovery studies in this species.

A Comparison of Blood Plasma Small Extracellular Vesicle Enrichment Strategies for Proteomic Analysis.

Proteomic analysis of small extracellular vesicles (sEVs) poses a significant challenge. A 'gold-standard' method for plasma sEV enrichment for downstream proteomic analysis is yet to be established. Methods were evaluated for their capacity to successfully isolate and enrich sEVs from plasma, minimise the presence of highly abundant plasma proteins, and result in the optimum representation of sEV proteins by liquid chromatography tandem mass spectrometry. Plasma from four cattle (Bos taurus) of similar physical attributes and genetics were used. Three methods of sEV enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These methods were combined to create four groups for methodological evaluation: UC + SEC, UC + SEC + UF, SEC + UC and SEC + UF. The UC + SEC method yielded the highest number of protein identifications (IDs). The SEC + UC method reduced plasma protein IDs compared to the other methods, but also resulted in the lowest number of protein IDs overall. The UC + SEC + UF method decreased sEV protein ID, particle number, mean and mode particle size, particle yield, and did not improve purity compared to the UC + SEC method. In this study, the UC + SEC method was the best method for sEV protein ID, purity, and overall particle yield. Our data suggest that the method and sequence of sEV enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.