Identification of gravity‐responsive serum proteins in spaceflight mice using a quantitative proteomic approach with data‐independent acquisition mass spectrometry

Physical inactivity associated with gravity unloading, such as microgravity during spaceflight and hindlimb unloading (HU), can cause various physiological changes. In this study, we attempted to identify serum proteins whose levels fluctuated in response to gravity unloading. First, we quantitatively assessed changes in the serum proteome profiles of spaceflight mice using mass spectrometry with data‐independent acquisition. The serum levels of several proteins involved in the responses to estrogen and glucocorticoid, blood vessel maturation, osteoblast differentiation, and ossification were changed by microgravity exposure. Furthermore, a collective evaluation of serum proteomic data from spaceflight and HU mice identified 30 serum proteins, including Mmp2, Igfbp2, Tnc, Cdh5, and Pmel, whose levels varied to a similar extent in both gravity unloading models. These changes in serum levels could be involved in the physiological changes induced by gravity unloading. A collective evaluation of serum, femur, and soleus muscle proteome data of spaceflight mice also showed 24 serum proteins, including Igfbp5, Igfbp3, and Postn, whose levels could be associated with biological changes induced by microgravity. This study examined serum proteome profiles in response to gravity unloading, and may help deepen our understanding of microgravity adaptation mechanisms during prolonged spaceflight missions.


INTRODUCTION
Prolonged spaceflight causes various physiological changes due to several environmental factors, including microgravity (μ-g), space radiation, and the stresses associated with a confined environment [1,2].Of these, μ-g exposure has the greatest impact on the body, and its physiological effects have been investigated using proteomic approaches in microorganisms, plants, and mammalian cells [3].Recent transcriptome analyses of skeletal muscle in mice have also shown microgravity-induced gene networks [4][5][6].In addition, various types of samples, such as stool, urine, plasma, and immune cells, were obtained from a pair of male monozygotic twins (one spent 340 days aboard the International Space Station (ISS), while another remained on Earth); these were analyzed using a wide battery of methods, resulting in telomeric, transcriptomic, epigenetic, proteomic, metabolomic, and immune data that gave rise to integrated, longitudinal, multidimensional descriptions of the effects of a prolonged spaceflight mission [7,8].These critical projects may accelerate the elucidation of the mechanisms whereby organisms adapt to gravitational stimuli.However, much remains unknown about which molecules are involved in the systemic response mechanisms to gravitational unloading during spaceflight.
Proteins play an essential role in every important component of organisms, and execute the functions encoded in the genome [9,10].
Proteins secreted by a wide variety of cells and tissues into the extracellular space are known as the secretome, and they function as endogenous regulators of physiological functions.Damage-associated molecular patterns (DAMPs), which are molecules released after cellular damage or stress, also regulate the activation of the immune system [11][12][13].Blood flows continuously throughout the body, thereby serving as the most fundamental means of transporting various proteins, including the secretome and DAMPs.Accordingly, changes in blood protein levels often reflect physiological and pathological conditions in vivo.Mass spectrometry (MS)-based quantitative proteomics using serum, which is the liquid fraction of whole blood, is therefore a rapidly advancing technique for addressing various challenges and identifying diagnostic markers [9].Hence, a comprehensive proteomic analysis of the serum, including characterizing the body's response to gravity unloading, should help characterize gravity adaptation mechanisms.
To this end, the quantitative analysis of proteins in plasma samples collected from cosmonauts or astronauts aboard the ISS has been performed using targeted MS and high-resolution MS-based multidimensional protein identification technology (MudPIT) [8,14,15].The results revealed protein responses associated with several biological processes during long-duration spaceflight, including innate immunity, lipid metabolism, and the coagulation cascade.However, cosmonauts are affected not only by μ-g exposure, but also by other factors such as space radiation, high carbon dioxide concentrations, and gravitational reloading associated with atmospheric re-entry and landing on Earth, making it difficult to identify specific gravity-responsive proteins in the human blood.
Hindlimb unloading (HU), a ground-based model of μ-g exposure, has also been used to examine changes in the musculoskeletal system,

Significance Statement
We aimed to identify serum proteins related to mechanisms of adaptation to gravitational stimuli.This study used serum samples collected from mice that all exhibited the same char- such as muscle atrophy and bone loss, in response to gravity unloading.It has provided mechanistic insights into physiological changes similar to those seen in the space environment, including cardiovascular changes, skeletal muscle atrophy, and bone loss [16].However, HU model mice are not exposed to systemic gravity unloading and therefore differ from spaceflight mice in terms of continuous gravitational loading of the forelimbs, fluid shifts, and discrepancies in stress responses between individuals [16].Therefore, the Japan Aerospace Exploration Agency (JAXA) has developed the Multiple Artificial Gravity Research System (MARS) for breeding mice under artificial 1-g (A1-g) conditions via centrifugal separation aboard the ISS [17].MARS allows us to compare the biological effects of μ-g and A1-g on mice raised in different gravity environments in space, making it possible to evaluate the systemic effects of physical inactivity [17,18].The effects of μ-g exposure on the retina [19], thymus [20], femur [21], and skeletal muscle [22,23], but not serum, of spaceflight mice bred using MARS have already been analyzed using proteomic and transcriptomic approaches, which showed that protein and gene expression of these tissues were influenced by gravitational unloading.On the other hand, our previous MS-based proteomic analysis of spaceflight mice could not rule out the possibility that gravity reloading after return to Earth affected protein expression in the soleus muscle [22].When evaluating the results of HU or spaceflight mice individually, we thus hypothesized that each nongravity stressor may make it difficult to identify systemic serum proteins that respond to gravity unloading.
Here, we used the serum of mice raised in different gravity environments on the ISS to identify serum proteins closely associated with each environment, as shown in the flowchart in Figure 1.We first constructed a customized mouse serum spectral library and established a quantitative analysis system for the mouse serum proteome using MS with data-independent acquisition (DIA-MS) [24].Using this system, we obtained the serum proteome profiles of mice exposed to μ-g and A1-g environments, as well as those of control and HU mice.Furthermore, comparing serum proteomic data between spaceflight and HU mice allowed for the identification of serum proteins that exhibit physiological changes induced by gravity unloading.We also collectively evaluated proteomic data from the serum, femur [21], and soleus muscle [22,23] of spaceflight mice.This study will help elucidate the adaptation mechanisms induced by gravitational stimuli during prolonged spaceflight missions.

Experimental animals sample collection
All experimental procedures were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" of the Japanese Physiological Society, the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health, and the ARRIVE guidelines.The details of spaceflight mouse experiments were previously described [18,22].Briefly, the mice were randomly divided into μ-g (n = 6) and A1-g (n = 6) groups and raised in separate MARS habitat cages aboard the ISS under each gravitational condition for 30 days.
Mice were further classified according to whether or not they consumed a diet containing 5% prebiotic fructo-oligosaccharides (FOS) (n = 3 per group), for the purpose of assessing the effects of FOS intake on the gut microbiome and immune cells [25].After spaceflight, mice were returned to Earth alive, followed by serum collection

Sample preparation for quantitative proteomic analysis
After the immunodepletion of albumin, IgG, and transferrin (approximately 80% of total protein in the blood) using a Mouse 3 Multiple Affinity Removal System column (Agilent Technologies, Santa Clara, CA, USA), mouse serum was precipitated by four volumes of cold acetone and redissolved in 50 mM ammonium bicarbonate (NH 4 HCO 3 ) containing 4 M urea.Subsequently, proteins were reduced with DTT (final concentration of 10 mM) and alkylated with 2-iodoacetamide (final concentration of 25 mM).The protein solutions were then diluted with 50 mM NH 4 HCO 3 (final concentration of 2 M urea) and incubated with trypsin at 37 • C for 16 h.The resultant peptides were desalted using a StageTip [27], and the subsequently eluted peptides were completely lyophilized and kept at −80 • C until use.

MS in the quantitative proteomic analysis
All experimental procedures were performed using a LC-MS system, with slight modifications to a previously reported serum proteomic analysis [28].For DIA-MS analysis of serum from spaceflight and HU mice, a customized spectral library was constructed using serum derived from control male C57BL/6J mice.Specifically, a total of 64 data-dependent acquisition (DDA) measurements were obtained using samples prepared through the following three fractionation methods: reversed-phase column [28].Tryptic-digested peptides were analyzed using a Q Exactive™ mass spectrometer coupled with an UltiMate™ 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA).Solvent A was 0.1% formic acid in 2% acetonitrile, and solvent B was 0.1% formic acid in 95% acetonitrile.Peptides were eluted using a gradient from 2% B for 0-5 min, 2% to 33% B for 5-180 min, and 95% B for 10 min, and then equilibrated for 20 min at 2% B. To create the spectral library for mouse serum proteome analysis, DDA mode analytical conditions consisted of a full MS1 scan with a resolution of 140,000 and a scan range from 350 to 1500 m/z, with the automatic gain control (AGC) target value set to 3e 6 (Full MS) or 5e 5 (MS/MS).Creation of the spectral library was performed using Spectronaut Pulsar X (Ver.12.0.2;Biognosys, Zurich, Switzerland) by searching against the iRT fasta database (Biognosys) and mouse protein sequences from the UniProtKB/Swiss-Prot database (version April 2021).Search results were filtered to satisfy a false discovery rate of 1% for both precursors and proteins, using the Spectronaut Pulsar X for identification.
For the quantitative proteomic approach, DIA mode analytical conditions consisted of a full MS1 scan with a resolution of 140,000 and a scan range from 400 to 1,220 m/z, with the AGC target value set to 5e 6 (Full MS), followed by 19 DIA variable windows acquired at a resolution of 70,000, with the AGC target value set to 3e 6 .The normalized collision energy was set to 27.DIA data were analyzed using the Spectronaut Pulsar X to identify and quantify proteins in the serum of spaceflight and HU mice using a customized spectral library as described in a previous report [28].Downstream statistical quantitative analysis was performed using Perseus software (ver.1.6.2.2; Max Planck Institute of Biochemistry) [28,29].All settings were set to default with slight modifications.Briefly, the samples were categorized into two groups, µspecifically g and A1-g groups in the spaceflight mouse experiment, and HU and control groups in the HU mouse experiment.The intensity values were log 2 -transformed, and only proteins present in at least 70% of samples in each group were used for further analysis [28].Statistical evaluation for proteomic analysis was also performed using the two-tailed Student's t-test.Differences Gene ontology (GO) enrichment analysis of biological processes was conducted using the functional annotation tool in DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/)[30]."Mus musculus" was selected as the background and analysis was performed by searching against the gene list in the original knowledge database for each software.The threshold for gene enrichment in annotation terms was determined using the Benjamini-Hochberg procedure with a p value < 0.05.All settings were set to default.mRNA expression levels in eight tissues (brain, colon, kidney, liver, lung, skeletal muscle, spleen, and testis) from C57BL/6 mice were examined using ExpressionAtlas (https://www.ebi.ac.uk/gxa/home) [31].

Discovery of mouse serum proteins differentially expressed in response to changes in gravity loading during spaceflight
To identify potential alterations of serum proteins in spaceflight mice exposed to gravity unloading, we used quantitative proteome analysis with DIA-MS (Figure 1) to evaluate serum collected from mice raised under a μ-g or A1-g environment aboard the ISS.A customized spectral library containing information on 1,106 mouse serum proteins was generated for use in this study.DIA-MS with this spectral library identified 568 proteins in serum samples from 12 spaceflight mice, and 515 proteins were selected for further statistical analysis by filtering based on valid values using Perseus software (Table S1).PCA suggested that gravity differences distinguished the sample groups more obviously than the presence or absence of FOS intake (Figure S1A).Although FOSs have been shown to increase bone mass in growing rats [25], FOS intake did not affect bone mass [21] or the absolute wet weight of the soleus or extensor digitorum longus muscle [22] in the spaceflight mice used in our experiment.Therefore, in this study, we compared serum proteome profiles between μ-g and A1-g environments without distinguishing between FOS intake.In an analysis using Perseus software, the threshold criterion for increased or decreased protein levels following μ-g exposure was set at p value [−log 10 ] > 1.3.Consequently, 34 and 72 proteins with increased or decreased serum levels were identified as being closely associated with gravity unloading (Table S1).

Biological functions of gravity-responsive proteins in serum from spaceflight mice
To determine biological functions, we performed functional annotation analyses of 106 protein sets expressed differentially in the serum of spaceflight mice.GO enrichment analysis performed with DAVID was used to assess the 106 protein sets with different levels after gravity unloading, and suggested that several of the serum proteins are involved in processes such as cell adhesion, extracellular matrix organization, mechanical stimulus response, fibrinolysis, responses to estrogen and glucocorticoids, osteoblast differentiation, ossification, and blood vessel maturation (Table S2).Estrogen and glucocorticoids are major causes of bone loss and skeletal muscle atrophy [32][33][34][35], and decreases in the serum levels of related gravityresponsive secreted proteins, namely growth/differentiation factor 8 (Mstn), 72 kDa type IV collagenase (Mmp2), and insulin-like growth factor-binding protein 2 (Igfbp2), may be involved in the physiological changes induced by microgravity exposure.Proteome profile changes in the serum of mice with microgravity exposure were also predicted by functional analysis within the IPA framework to be involved in the  S3).Fibroblasts not only build connective tissue, but also serve as precursors to specialized mesenchymal cells, such as bone-forming osteoblasts [36].A decrease in fibroblast proliferation might therefore disrupt the bone remodeling system that is normally maintained by balanced cellular activity between osteoblasts and osteoclasts.Therefore, our results suggest that the serum proteome of mice reflects the mechanism of bone loss under gravity unloading during spaceflight.However, deficiency of the aforementioned Mmp2 has been associated with reduced bone mineral density and bone fragility [37,38].Mmp2 is also involved in ECM remodeling of skeletal muscle and its inactivity leads to reduced muscle contractility [39].Mstn (myostatin), a myokine that is produced and released by muscles throughout the body, also acts as a negative regulator of skeletal-muscle growth [40], and animals either lacking Mstn or treated with an Mstn inhibitor have significantly more muscle mass than controls [41].Mice with Igfbp2 knockout, which inhibits bone development, are protected against trabecular bone loss [42].Many of these proteins contribute to a variety of other biological functions, and it remains unclear why changes in their serum levels occur in mice with gravity unloading exposure.

Identification of mouse serum proteins closely responsive to gravity change
We next performed a quantitative proteome analysis with DIA-MS to compare the serum proteome profile of HU mice, a ground-based mouse model of gravity unloading [16], with that of control mice.A total of 563 proteins were identified using the spectral library customized for this study, and 493 were selected for further statistical analysis by filtering based on valid values using Perseus software.Of these, 19 and 148 proteins with increased or decreased levels, respectively, were identified as being significantly associated with HU (p value [−log 10 ] > 1.3) (Table S4).GO enrichment analysis with DAVID and functional analysis with IPA were used to evaluate 167 protein sets expressed differentially in the serum of HU mice.As in spaceflight mice, some of the proteins were associated with biological processes such as cell adhesion, fibrinolysis, extracellular matrix organization, and responses to mechanical stimuli (Table S5), and contributed to the downregulation of both fibroblast proliferation and connective tissue cell differentiation (Table S3).Both spaceflight and HU mice exhibit muscle atrophy and bone loss of the hindlimbs, but serum proteome profiles are also affected by factors other than physical inactivity associated with gravity unloading, such as fluid shifts and gravitational loading of the forelimbs [16].To identify mouse serum proteins associated with changes in these common biological processes, we further compared proteins with differential serum level between the spaceflight and HU experiments.Of the 448 proteins (80.0% of the total 560, Table S6) commonly identified in the two experiments, 30 proteins, including Mmp2, Igfbp2, tenascin (Tnc), cadherin-5 (Cdh5), and melanocyte protein PMEL (Pmel), showed similar serum level changes associated with gravity unloading in both spaceflight and HU mice (Table 1).Most of these are secreted or transmembrane proteins, many of which are strongly expressed in the liver, lung, and spleen (Table S7).Among them, the expression levels of Tnc are known to be strongly induced by mechanical stimuli such as load-induced bone remodeling or muscle overload [43], and Tnc inhibition markedly suppresses bone formation and bone mineralization [44,45].Cdh5 is a transmembrane adhesion protein that localizes to the endothelial cell junction and is central to a variety of cellular processes, including regulation of endothelial permeability, angiogenesis, and remodeling [46,47].Mechanotransduction by the Cdh5 complex in vascular endothelial cells is also known to cause local cytoskeletal remodeling, alter peripheral cell-cell junctions, and activate global signals that disrupt intercellular contacts [48].Pmel is a type I single transmembrane glycoprotein that is expressed in the substantia nigra, pigmented melanocytes, and retinal pigment epithelium, and it also TA B L E 1 List of proteins with similar serum level changes (p value [-log 10 ] > 1.3) in spaceflight mice with μ-g versus A1-g exposure and in HU versus control mice.has an interesting role as a functional amyloid [49,50].Although no direct relationship between Pmel and gravitational response has been reported, it was shown that amyloid fibrils with a unique shape, different from those on the ground, formed in a μ-g environment [51].

Gene
These serum protein profiles could therefore represent physiological changes induced by gravity unloading in both spaceflight and HU mice.
These results also suggest that the analysis of serum proteome profiles may provide insight into what physiological changes may occur during prolonged spaceflight missions.

Identification of bone-and muscle-related mouse serum proteins responsive to gravity unloading during spaceflight
In a previous study, we obtained proteomic data for the femur [21] and soleus muscle [22,23] of the same spaceflight mice as those used in this study.To ascertain the relationships between protein level changes in serum, femur, and soleus muscle during prolonged spaceflight missions, we collectively evaluated the proteomic data from all three sources in TA B L E 2 List of proteins with similar level changes (p value [-log 10 ] > 1.3) in the serum, soleus muscle, and femur of spaceflight mice with μ-g versus A1-g exposure.2).Most of these were secreted proteins, many of which were strongly expressed in the liver and skeletal muscle (Table S7).The STRING tool (https://stringdb.org/), which is used for network analysis of protein-protein interactions, showed that most of these proteins may be related to each other (Figure S2).Among these, the serum levels of the secreted proteins, Igfbp3 and Igfbp5, which were similarly increased in the serum and femur by gravity unloading during spaceflight, are known to be related to negative regulation of osteoblast differentiation [52,53].

Serum
Insulin-like growth factors (IGFs) play a key role in normal skeletal development and bone remodeling and IGFBPs regulate the activity of IGFs.In fact, serum levels of Igf1 were increased following exposure to microgravity (Table S1).However, the previous study [54] also demonstrated that some functions of IGFBPs inhibit IGF action, whereas others enhance it.IGFBPs have also been shown to modulate other biological processes independently of their ability to bind IGFs.
Accordingly, it may be difficult to explain the effects of increased Igfb3 and Igfb5 levels in the serum and bone of spaceflight mice.In addition, periostin (Postn), which is a secreted protein that was similarly decreased in the serum and soleus muscle by gravity unloading during spaceflight, is known to promote collagen cross-linking by supporting bone morphogenetic protein 1 (BMP1) on the extracellular matrix [55].
Postn has also been reported to contribute to osteogenesis and differentiation in response to mechanical stress [56].Furthermore, serum POSTN levels are associated with the prevalence of knee osteoarthritis and the risk of osteoarthritis development and progression in women [57].Recently, increasing basic and clinical data have suggested that skeletal muscle and bone are both spatially and metabolically connected [58][59][60], and the proteins discussed above, including POSTN, may be serum indicators for physiological changes associated with gravity during spaceflight, such as bone loss and muscle atrophy.
This study, however, has some limitations, particularly the difficulty in performing verification experiments due to the lack of sufficient specimens from spaceflight mice.An additional limitation was the inability to collect mouse serum samples during spaceflight due to operational issues.Therefore, spaceflight mice, which were returned to the ground alive, had to be retrieved at sea and transported to the dissection site, which took 2 days.The present study thus had to consider that the serum proteome may have been influenced by reloading after return to Earth.

CONCLUDING REMARKS
In summary, we used DIA-MS to perform a comprehensive quantita- acteristics of hindlimb bone loss and muscular atrophy, but were raised under different environments: spaceflight or hindlimb unloading (HU).Comprehensive mouse serum proteome analysis using mass spectrometry may provide insight into the physiological changes that occur during prolonged spaceflight missions.Furthermore, collectively evaluating the proteomic data from the serum of spaceflight and HU mice and those from the serum, femur, and soleus muscle of spaceflight mice may identify the serum proteins involved in the changes induced by gravity unloading.Our study represents a first step toward elucidating the adaptation mechanisms induced by gravity unloading during prolonged spaceflight missions.

within 2
days after landing at a ground-based laboratory in the United States.The HU mouse experiments were conducted with the approval of the Committee on Animal Care and Use of Yokohama City University (accreditation no.: F-A-16-066).These experiments were conducted on the basis of a previous report[26] with slight modifications, and used male C57BL/6J mice (9−10 weeks old; Chubu Kagaku Shizai, Nagoya, Japan) that were randomly divided into the HU group (n = 8) and control group (n = 8).After anesthetization with 2% isoflurane, 2-0 sterile surgical steel wire was passed through the fifth intervertebral disc in the tail (counting from the end of body) and formed into a ring shape; the ring was then lifted to keep the hindlimbs off the cage floor.The control group consisted of mice with the same tail ring but without tail suspension.The mice in each cage were kept for 42 days and had free access to food and water throughout the study.In both spaceflight and HU mice, the collected venous blood was incubated at 22 • C for 30 min, then centrifuged at 3000 × g for 15 min at 22 • C to collect serum, and the serum was stored at −80 • C until use.
immunodepletion using a Mouse 3 Multiple Affinity Removal System column and fractionation into 20 fractions by a HPLC system coupled with a C4 reversed-phase column (Grace, Columbia, MD); immunodepletion and separation into 24 fractions based on molecular weight by SDS-PAGE; and protein enrichment using a Proteominer (Bio-Rad Laboratory, Hercules, CA) and fractionation into 20 fractions by a C4 with an unadjusted p value [−log 10 ] > 1.3 were considered statistically significant.Several variables, including food intake and individual characteristics, might influence serum proteome profiles.In the discovery studies, therefore, unadjusted p values were used to avoid missing biologically important and meaningful changes in serum protein levels.Functional annotation analyses were also performed by Ingenuity Pathway Analysis (IPA) (Content version: 84978992, Release Date: 2022-11-27; Qiagen, Hilden, Germany) using Fisher's exact test.

F I G U R E 2
Network of proteins determined by biological function analysis within the IPA framework.Dotted lines indicate indirect interactions.downregulation of both fibroblast proliferation and connective tissue cell differentiation (Figure 2 and Table tive proteomic analysis of serum samples collected from spaceflight and HU mice to identify serum proteins involved in changes induced by gravitational stimuli.The serum levels of 30 proteins, including Mmp2, Igfbp2, Tnc, Cdh5, and Pmel, showed similar variations in both models, which may reflect biological changes under gravity unloading.Collective evaluation of serum, femur, and soleus muscle proteome data from spaceflight mice also identified 24 proteins, including Igfbp5, Igfbp3, and Postn, whose levels changed similarly in the serum and the femur and/or soleus muscle in response to microgravity exposure.These changes may be related to physiological alterations, such as bone loss and muscle atrophy, induced by the lack of gravitational stimuli during extended spaceflight.This study is expected to lead to novel insights into the mechanisms of adaptation to the absence of gravitational stimulation during prolonged spaceflight missions.