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Targeted Mass Spectrometry for Serum Amyloid a (SAA) Isoform Profiling in Sequential Blood Samples from Experimentally Staphylococcus Aureus Infected Pigs

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Targeted Mass Spectrometry for Serum Amyloid a (SAA) Isoform Profiling in Sequential Blood Samples from Experimentally Staphylococcus Aureus Infected Pigs Downloaded from orbit.dtu.dk on: Sep 24, 2021 Targeted mass spectrometry for Serum Amyloid A (SAA) isoform profiling in sequential blood samples from experimentally Staphylococcus aureus infected pigs Leuchsenring, Anna Barslund; Karlsson, Christofer; Bundgaard, Louise; Malmström, Johan; Heegaard, Peter M. H. Published in: Journal of Proteomics Link to article, DOI: 10.1016/j.jprot.2020.103904 Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Leuchsenring, A. B., Karlsson, C., Bundgaard, L., Malmström, J., & Heegaard, P. M. H. (2020). Targeted mass spectrometry for Serum Amyloid A (SAA) isoform profiling in sequential blood samples from experimentally Staphylococcus aureus infected pigs. Journal of Proteomics, 227, [103904]. https://doi.org/10.1016/j.jprot.2020.103904 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Journal of Proteomics 227 (2020) 103904 Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot Targeted mass spectrometry for Serum Amyloid A (SAA) isoform profiling in sequential blood samples from experimentally Staphylococcus aureus infected T pigs Anna Barslund Leuchsenringa, Christofer Karlssonb, Louise Bundgaarda, Johan Malmströmb, ⁎ Peter M.H. Heegaarda, a Department of Biotechnology and Biomedicine, DTU Bioengineering, Technical University of Denmark, Lyngby, Denmark b Department of Clinical Sciences, Lund, Division of Infection Medicine, Lund University, BMC, Lund, Sweden ABSTRACT Serum amyloid A (SAA) is a well-described acute phase protein induced during the acute phase response (APR) to infection. Four isoform specific genes are found in most mammals. Depending on species, SAA3 and SAA4 are generally preferentially expressed extrahepatically whereas SAA1 and SAA2 are hepatic isoforms dominating the SAA serum pool. Little is known about how specific infections affect the serum SAA isoform profile, as SAA isoform discriminating antibodies are not generally available. An antibody independent, quantitative targeted MS method (Selected Reaction Monitoring, SRM) based on available information on porcine SAA isoform genes was developed and used to profile SAA in serum samples from pigs experimentally infected with Staphylococcus aureus (Sa). While results suggest SAA2 as the main circulating porcine SAA isoform, induced around 10 times compared to non-infected controls, total SAA serum concentrations reached only around 4 μg/ mL, much lower than established previously by immunoassays. This might suggest that SAA isoform variants not detected by the SRM method might be present in porcine serum. The assay allows monitoring host responses to experimental infections, infectious diseases and inflammation states in the pig at an unprecedented level of detail. It can also be used in a non-calibrated (relative quantification) format. Significance: We developed an SRM MS method which for the first time allowed the specific quantification of each of the circulating porcine SAA isoforms (SAA2, SAA3, SAA4). It was found that SAA2 is the dominating circulating isoform of SAA in the pig and that, during the acute phase response to Sa infection SAA2, SAA3 and SAA4 are induced approx. 10, 15 and 2 times, respectively. Absolute levels of the isoforms as determined by SRM MS were much lower than reported previously for total SAA quantified by immunosassays, suggesting the existence of hitherto non-described SAA variants. SRM MS holds great promise for the study of the basic biology of SAA isoforms with the potential to study an even broader range of SAA variants. 1. Introduction specific isoform of SAA (SAA1–4), have been observed in most species; all genes having the same four-exon three-intron structure [9,12]. The Tissue perturbation, including infection and inflammation is ac- four SAA genes are highly conserved throughout evolution, and this, companied by a generalized set of immediate host reactions at the together with the rapid and huge APR of SAA proteins, indicate that tissue site as well as by an array of systemic reactions known as the SAA and its isoforms most probably have important functions during acute phase response (APR) [1,2]. The APR leads to major changes in the response to tissue perturbations [13]. circulating concentrations of a subset of serum proteins called acute Still, the precise function of SAA and its role during the APR have phase proteins [1,3]. The acute phase proteins constitute a set of very not been established [14,15]. It is speculated that SAA plays an im- useful inflammation biomarkers, but they have low discriminative portant role during the early phases of inflammation [16,17], linking power as the type of inflammation/infection has a limited effect on the complex network of cells (macrophages, hepatocytes and others) their acute phase response kinetics and magnitude [4–6]. and proteins mediating inflammation together, functioning as a cyto- Serum amyloid A (SAA) is an important and well-described highly kine-like protein in cell-cell communication as well as feedback in regulated acute phase protein in vertebrates [7,8]. It is a small apoli- pathways that are immunologic, neoplastic, and protective during in- poprotein [9–11], present in the blood circulation at low levels (a few flammation [18]. Well-described characteristics include the ability of μg/mL or lower in most species) at homeostasis, however, may be in- circulating SAA protein to bind to high-density lipoprotein, replacing duced several hundredfold during the APR in most vertebrates, in- apolipoprotein A1 during the APR, becoming the predominant apoli- cluding pigs [10]. Four very similar SAA genes, each coding for a poprotein of high-density lipoprotein [9,11]. SAA can directly induce ⁎ Corresponding author. E-mail address: [email protected] (P.M.H. Heegaard). https://doi.org/10.1016/j.jprot.2020.103904 Received 24 April 2020; Received in revised form 19 June 2020; Accepted 29 June 2020 Available online 21 July 2020 1874-3919/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). A.B. Leuchsenring, et al. Journal of Proteomics 227 (2020) 103904 pro-inflammatory cytokines and chemokines in monocytes and granu- complex biological samples such as serum [30–32]. SRM exploits the locytes, and it has chemotactic effects on various cell types through unique capability of triple quadrupole (QQQ) mass spectrometers to binding to formyl-peptide receptor-2 (FPR2) and Toll-like receptor-2 filter out unrelated ions both at the tryptic peptide stage and at the (TLR2) [19]. In the latter work the efficacy towards the two types of fragment ion stage according to preselected mass to charge ratios receptors mentioned was furthermore demonstrated to differ between [30–32]. We developed a quantitative, antibody-free SRM-MS method isoforms of human SAA (SAA1 isoform variants and SAA2) [19]. Most for quantifying specific circulating porcine SAA isoforms directly in recently, cytokine-like properties have been ascribed to SAA as it seems serum samples from pigs infected experimentally with Sa. This allowed to be intimately implicated in the intestinal control of Th17 immunity, us to correlate tissue specific expression of SAA isoforms (mRNA) with SAA itself being directly induced in epithelial cells of mucosal tissue by SAA isoforms (protein) in the circulation. This is of interest not only for IL22, which can be controlled by the microbiota in the absence of in- elucidating possible specific biological functions of SAA isoforms, but flammation [20]. also for describing the possible utility of SAA isoforms as differential, The predominant site for synthesis of SAA is the liver [21,22]; circulating biomarkers of infection with the potential to differentiate however, extrahepatic synthesis has repeatedly been demonstrated inflammatory responses having infectious (e.g. bacterial versus viral), [14,23]. Generally, SAA1 and SAA2 are preferentially expressed in the autoimmune, neurodegenerative and metabolic origins, respectively. liver, while SAA3 is found to be expressed in both hepatic and non- hepatic tissues during an APR [15]. In the pig Olsen et al. [15] studied 2. Materials and methods the relative expression of SAA1, SAA2, SAA3 and SAA4 genes in spleen, liver and lung tissue from pigs experimentally infected with the swine 2.1. Ethics statement specific pathogen Actinobacillus pleuropneumoniae (Ap), and a porcine strain of Staphylococcus aureus (Sa), respectively, using SAA isoform All animal procedures were conducted according to protocols ap- specific quantitative (q) PCR assays. Results showed that for both in- proved by the Danish Animal Experiments Inspectorate (License fections, increased hepatic expressions of SAA2,
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