Proteograph™-based proteome and sphingolipidome analyses identified novel serum biomarkers to monitor astronauts' health in spaceflight

Introduction Long duration spaceflight leads to significant muscle mass and strength loss, which current inflight countermeasures can only partially mitigate. This study aimed to identify as yet unexplored low-abundance serological biomarkers in astronaut blood samples as meaningful biological signatures for deeper insight into musculoskeletal adaptation as complementary protocol for upgraded health monitoring in future spaceflight.Methods Serum samples from eight long-duration mission (180 days and more) astronauts were collected pre-flight (baseline), in-flight (two time points) and post-flight (two time points). Pre- and 3/5 days post-flight samples were analyzed using the Proteograph (TM) XT kit, enabling the detection of protein-level differences. Mass spectrometry data were acquired in Data-Independent Acquisition mode, analyzed with Perseus using paired Student's t-tests, and then analyzed by Ingenuity Pathway Analysis to prioritize affected pathways. Targeted serum sphingolipids quantification was carried out across all time points using Multiple-Reaction Monitoring Mass Spectrometry. Immunoblotting was performed on serum and muscle extracts from a previous space flight mission, for selected proteins.Results Among 1,718 detected proteins, 153 showed significant changes in abundance, with 11 displaying marked alterations targeting osteogenesis (spondin, osteomodulin), lipid metabolism (perlipins 1, 3 and 4) and ECM remodelling (collagen alpha-2(XI) chain, collagen triple helix repeat containing 1). Perlipin 4, collagen alpha-2(XI) chain, and collagen triple helix repeat containing 1 were assessed by immunoblotting at all time points (pre/in/post-flight). Functional pathway analysis identified 10 pathways related to muscle function and cytoskeletal organization, and one (reelin, BDNF) associated with brain function. We also found changes in enzyme levels from pre-and post-flight muscle extracts that can be associated to decreased levels of total ceramides in the first in-flight sample (IF1), followed by a rebound in subsequent samples (IF2, POST1) showing increased glucosyl ceramide levels.Conclusion Long-duration spaceflight induces systemic and muscle-specific changes providing deeper insights into the multifaceted mechanisms of molecular pathways related to neuro-musculoskeletal adaptation to microgravity. Identification of low-abundance serum biomarkers from astronaut blood using high-resolution and precision protocols present as novel complementary tools for broader assessment of musculoskeletal health conditions in crewed future deep space missions.