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™ 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.