Going deep into the acute phase protein response

Tissue perturbation and infection are accompanied by a generalized set of immediate host reactions at the site of disturbance as well as an array of systemic reactions known as the acute phase response (APR). The APR leads to major changes in circulating concentrations of a subset of blood proteins called acute phase proteins. Serum amyloid A
(SAA) is a well-described acute phase protein in vertebrates synthesized in the liver and non-hepatic tissue. Most species have four genes each encoding a specific isoform of SAA, which have found to be differentially regulated depending on infection and tissue. This is a unique feature of SAA not found in other acute phase proteins and makes circulating SAA and its isoforms very interesting biomarker candidates. However, SAA isoform analysis is cumbersome, non-quantitative and no antibodies specific for SAA isoforms have been developed, making SAA isoform distribution at the protein level less characterized. Using mass spectrometry (MS)-based methods may overcome these problems as MS is more specific and adequately sensitive. In this Ph.D. project, the native composition and presence of SAA and its isoforms in pig serum was first investigated through 2D gel electrophoresis (2DE) and the MS-based method Matrix-assisted laser desorption/ionization (MALDI) with a time-of-flight (TOF) mass spectrometry. Subsequently a new targeted quantitative MS method was developed based on selected reaction monitoring (SRM) mass spectrometry. 2DE and MALDI-TOF confirmed the presence of an APR in the investigated serum samples, but only SAA2 and SAA4 could be identified. As this experimental set-up is both time demanding and not the most
suitable for biomarker development, the SRM method developed was used to discriminate and quantify the porcine SAA isoforms with high specificity and sensitivity in porcine serum samples from two separate and previously performed infection studies. For improved quantitative performance, isotopically labeled SAA peptides were used to calibrate the method. In Paper I, the SRM method was applied to sequential serum samples from a previous experiment in which pigs were infected with Staphylococcus aureus (Sa) where all circulating porcine SAA isoforms were detected. The SAA isoform profiles correlated with the total SAA response previously established by ELISA, and a close correlation between the relative hepatic SAA isoform mRNA abundance and SAA isoform profiles in the circulation was also found. This proved that antibody independent MS-based quantification is possible at high specificity and sensitivity allowing discrimination between a set of very similar circulating SAA variants directly in serum samples. The results unequivocally establish SAA2 as the main circulating porcine SAA isoform. Porcine serum samples from pigs infected with Actinobacillus pleuropneumoniae (Ap) in another previous experiment were also analyzed, and while all SAA isoforms could be detected, only relative SAA concentrations could be measured as the selected proteotypic peptides did not perform well enough to provide absolute quantification. An additional set of porcine serum samples from a previous experiment on Sa osteomyelitis in pigs were analyzed in which all SAA isoforms were detected and quantified. Both relative and absolute SAA concentrations were measured and correlated well with each other in this sample set. Relative and absolute SAA isoform concentrations were compared across the three sample sets, and each set showed a different SAA isoform composition supporting the concept that different types of infections - for example having different tropism as with Ap and Sa - will result in infection-specific SAA isoform profiles. The prospect of using SAA isoform profiling to achieve increased diagnostic power from acute phase protein biomarkers, and to discriminate between different infections and/or
infections with different tissue tropism, is of great importance. It would be very interesting to investigate further the potential of this approach for a detailed characterization of mass variants of circulating proteins and the possible links to their tissue origin.