Spaceflight (SF) is one of the most extreme conditions and is resembled by a complex of factors, such as microgravity, extreme acceleration and cosmic radiation, which affect many physiological changes in the human organism [1, 2]. The molecular mechanisms of adaptation to SF conditions are not fully investigated and are important to study at all stages of long-duration missions on the International Space Station (ISS) [3–5]. Due to some technical, logistical and economical limitations (including those caused by weightlessness), the identification of changes occurring in the organism onboard of the ISS remains a difficult task [6].
Multi-omics technologies may provide new information at the molecular level regarding organism adaptation to SF conditions including changes in expression profiles of DNA and RNA [7], concentration profiles of proteins and metabolites [8]. The NASA multi-omics project collected 317 samples from twin astronauts (one of them spent a year on the ISS, while the other remained on Earth), and identified proteins that could be associated with observed changes in vascular wall dimensions [9]. This study has paved the way for future multi-omics studies, including those at the single-cell level, and has provided the first comprehensive molecular profile of an astronaut [10]. The proper collection and storage of biomaterial during SF is an important aspect of aerospace medicine, and has been given significant emphasis in the frame of new Space Omics and Medical Atlas (SOMA) initiative [5].
One of the relevant methods available is the dried blood spots (DBS) micro-sampling technique which is useful for various research studies and wide-population screenings [11–13]. DBS micro-sampling acquires a very small amount of blood (~ 20µl) and can be self-collected via a non-invasive finger capillary puncture. The tool kit for this procedure is simple, small, and easy to use onboard. Sheets of Whatman paper used as DBS sample holders can be periodically delivered by transport shuttles to Earth for DBS analysis in the lab. DBS samples are stable under standard environmental conditions for many years and contain thousands of analytes that can be re-eluted for subsequent analysis [14].
Analysis of the human blood proteome is challenging because almost all proteins synthesized in the human body eventually turn up in plasma and the dynamic range of protein concentrations in it is about eleven orders of magnitude. Liquid chromatography coupled to mass spectrometry (LC-MS) is capable for reproducibly quantifying up to 700 proteins in DBS samples, with a concentration range that spans four orders of magnitude [12, 15, 16]. Targeted quantitative proteomic analysis revealed that the concentrations of 190 proteins were stable in DBS samples stored up to 2 months at ambient temperature [12]. Thus, DBS can be considered as a valuable object for monitoring physiological changes at the molecular level under extreme conditions and is available for collection even during SF as it is suitable for long-term storage at room temperature.
A deep blood proteome analysis can facilitate the discovery of new data and mechanisms of human adaptive response to SF conditions. Mass-spectrometry-based proteomic techniques allow to detect alterations caused by SF. In a recent study initiated by NASA the first plasma samples for proteomic analysis were collected during SF from 4 astronauts [5]. Multidimensional Protein Identification Technology (MudPIT) was successfully applied and revealed 19 proteins whose expression was significantly altered due to SF. Further, GO analysis revealed that these proteins are involved in inflammatory responses, the cytoskeleton system, and metabolism that potentially have functional roles in response to SF or re-adaptation to Earth environment [4]. Our group has previously demonstrated changes in the proteome composition after long-duration (169–199 days) missions on the ISS, but only samples collected before and after SF were analyzed [17, 18]. As spaceflight becomes more common with commercial crews, blood-based measures become actual for health monitoring during short-duration spaceflight and the first study already performed within the SpaceX Inspiration4 (i4) mission [19]
The aim of the current study was to apply the method of blood proteome analysis with DBS micro-sampling technique to study the physiological response to SF conditions at the molecular level. Targeted mass-spectrometry based proteomics with a validated assay of stable isotope-labeled peptide standard (SIS) for 119 blood proteins was selected as a robust and precise tool for quantitative analysis. We considered the proteins and corresponding peptide panel from the BAK270 MRM assay [20]. The assay was developed for analysis of potential protein biomarkers for cardiovascular diseases, including 61 FDA-approved biomarkers in blood plasma. The robustness of the MRM assay for selected blood proteins was recently demonstrated [21]. Finally, a targeted proteomic analysis of 58 DBS samples collected from 7 cosmonauts during long-duration SF (169–199 days) was performed. To our knowledge this is the first DBS proteomic study for monitoring the adaptive reactions of cosmonauts during long-term SF.