Targeted mass spectrometry for Serum Amyloid A (SAA) isoform profiling in sequential blood samples from...

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.