Multiplex mass spectrometry-based analysis of inflammatory proteins and their isoforms in serum samples of analog astronauts

Inflammation is a complex mechanism of host defense response to stimulus (e.g. infection, tissue damage, or cancer development), tissue repair and recovery. Activated cells of the immune system release signal proteins that activate systemic response via translation of acute phase proteins (APPs). The quantification of selected highly conserved APPs, such as C-reactive protein (CRP), α-1 antitrypsin (A1AT), serum amyloid A (SAA), and α-1-acid glycoprotein (A1AG), calprotectin, haptoglobin (HP) and adaptive immunity effectors (i.e., immunoglobulins, myeloperoxidase) presumably allows for stratification among specific types of inflammation, and for differentiation between physiological and pathological responses.

APPs are routinely analyzed in clinics using high throughput immunoassays based on immunochemical interaction between protein and antigen. However, immunoassays rarely quantify isoforms of APPs. Although the function of isoforms is not known, the quantification of individual isoforms may help to characterize state of inflammation.

We have developed a multiplex mass spectrometry method for the quantification of APPs, adaptive immunity effectors and apolipoproteins in serum samples, including exact protein isoforms (i.e. SAA1, SAA2, CRP1, A1AT1), and to detect single nucleotide polymorphism (SNP).

The method has been applied to serum samples of 6 analog astronauts. Samples were collected before and after 6-day long APICES (Astroland Project Inside Caves for Earth-based Space exploration) Space Analogue Mission, studying the effect of space travel on astronaut"s health. All analog astronauts showed low concentration of APPs before and after the mission. A slight decrease in Apolipoprotein A1 was observed after the mission. Although the samples were "blind" for analysis, the quantification of IgG isoforms meant the specific isoform ratio per astronaut was able to be identified, and samples paired. Furthermore, two analog astronauts showed a polymorphism in haptoglobin sequence TEGDGVYTLNNEK, possibly indicating haptoglobin phenotype Hp2-2 (missing a1 unit HP).