Peptidome Analysis of Umbilical Cord Mesenchymal Stem Cell (hUC-MSC) Conditioned Medium from Preterm and Term Infants

DOI: https://doi.org/10.21203/rs.3.rs-28770/v1

Abstract

Background : The therapeutic role of mesenchymal stem cells (MSCs) has been widely confirmed in several animal models of premature infant diseases. Micromolecule peptides have shown promise for the treatment of premature infant diseases. However, the potential role of peptides secreted from MSCs has not been studied. The purpose of this study is to broaden the knowledge of the hUC-MSC secretome at the peptide level through peptidomic profile analysis. Methods : We used tandem mass tag (TMT) labeling technology followed by tandem mass spectrometry to compare the peptidomic profile of preterm and term umbilical cord MSC (hUC-MSC) conditioned medium (CM). Gene Ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA) were conducted to explore the differentially expressed peptides by predicting the functions of their precursor proteins. And we investigated the effects of differentially expressed peptides on human lung epithelial cells stimulated by hydrogen peroxide (H 2 O 2 ). Results : A total of 131 peptides derived from 106 precursor proteins were differentially expressed in the preterm hUC-MSC CM compared with the term group, comprising 37 up-regulated peptides and 94 down-regulated peptides. Bioinformatics analysis showed that these differentially expressed peptides may be associated with developmental disorders, inflammatory response and organismal injury. We also found that two differentially expressed peptides reduced the levels of TNFα and IL 1β mRNA in  H 2 O 2  -treated human lung epithelial cells. Conclusions : In summary, this study provides further secretomics information on hUC-MSCs and provides a series of peptides that might have roles in premature infant diseases, which may be helpful for prevention and treatment.

1. Introduction

The incidence of preterm birth has increased over the past 20 years in most countries [1, 2]. Despite recent advances in perinatal medicine, severe diseases related to premature birth, including periventricular leukomalacia (PVL), bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC) and retinopathy of prematurity (ROP), remain major causes of mortality and morbidity, which represent a heavy burden for families and society [3]. Therefore, it is an urgent and significant task to develop new safe and effective treatments to improve the prognosis of these diseases in premature infants.

In the past several decades, the development of mesenchymal stem cell (MSC) therapy and its continuous advancement have gained extensive attention. MSCs are multipotent progenitor cells, which can be raised from different tissues, for instance, adipose tissue, umbilical cord and bone marrow [4, 5]. Human umbilical cord MSCs (hUC-MSCs) are easily accessible and can be harvested from donors without risks or damage [6]. Additionally, the therapeutic application of MSCs is not limited by the aging-like nature of adult tissues such as bone marrow and adipose tissue [7, 8]. Mechanically, MSCs function in vivo via direct differentiation or paracrine action. The therapeutic potential of MSC engraftment has been proved in premature infant diseases, and early clinical trials in preterm neonates with BPD (NCT01297205 [9], NCT01632475 [10]) and severe intraventricular hemorrhage (NCT02274428 [11]) have been conducted. A myriad of bioactive factors are readily available in the conditioned medium (CM) of MSCs and the medium can mediate multiple known functions of MSCs, such as angiogenesis, anti-fibrosis and anti-inflammatory effects [12]. Extracellular vesicles such as exosomes have been isolated from CM, and they have been shown to contain microRNAs and proteins, which partially mediated the effects of MSC [13–15]. Many studies have established that the secretome from MSCs can reduce organ damage in animal models of PVL, BPD, NEC and ROP [16–20]. Other studies have also shown that soluble factors such as Angiopoietin-1 (Ang-1), Heme oxygenase-1 (HO-1) and Erythropoietin (EPO) may be principally responsible for the ability of MSC-CM to ameliorate inflammation, angiogenesis, fibrosis and so on [21–23]. More types of MSC secreted factors and regulatory mechanisms still need to be established.

Peptides, a type of compound with two or more amino acids connected by peptide bonds, have been shown to play important roles in the treatment of diseases. Glucagon-like peptide-1 (GLP-1), a well-known peptide hormone secreted from the L cells of the duodenum, colon, terminal ileum and rectal mucosa, has been used in the clinical treatment of type 2 diabetes [24]. Extrinsic calcitonin gene-related peptide (CGRP) could suppress apoptosis, oxidative stress and ROS production in hyperoxia-induced alveolar epithelium type II (AEC II) cells [25]. WKYMVm hexapeptide could attenuate hyperoxia-induced lung injuries in newborn mice [26]. Additionally, peptides from human milk such as PDC213, β-casein 197 and Casein201 exhibited obvious antimicrobial effects on the common pathogenic bacterial species S. aureus and Y. enterocolitica in neonatal intensive care units [27–29]. These studies indicated that peptides may hold great promise for the treatment of premature infant diseases. However, the secreted peptidomic profile from hUC-MSCs have not been fully characterized.

In the present study, we compared the secreted peptides from preterm and term hUC-MSCs using the tandem mass tag (TMT) labelling method with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. This study helps to broaden the knowledge of the hUC-MSC secretome at the peptide level through peptidomic profile analysis. Moreover, using Ingenuity Pathway Analysis (IPA) software, we predicted that the differentially expressed peptides are associated with developmental disorder, inflammatory response and organismal injury, indicating that they may be useful for treating premature respiratory diseases. And we preliminarily investigated the effect of differentially expressed peptides on human lung epithelial cells.

2. Materials And Methods

2.1 Patients and samples

Umbilical cords were obtained from six infants without genetic or structural anomalies delivered at 27–41 weeks of gestation with parental written consent. Cases involving maternal diabetes, pre-eclampsia, eclampsia, intrauterine growth retardation (IUGR) or infectious diseases were excluded, because these factors may influence cell proliferation, cytokine expression and other functions [30–34]. This study was approved by the Ethics Committee of Changzhou Maternal and Child Health Care Hospital (approval number: 2019126) and conducted in accordance with the approved guidelines.

2.2 Preparation of hUC-MSCs and CM

Human umbilical cords were collected after preterm (n = 3) or full term (n = 3) deliveries. hUC-MSCs were obtained by the tissue explants adherent method, as previously reported [17, 35]. Each umbilical cord (about 10 cm) was washed in phosphate-buffered saline (PBS; Gibco, Grand Island, CA, USA) and with 1% penicillin/streptomycin (P/S; Gibco) to remove residual blood from the vein and arteries. After the cord was cut longitudinally, and the arteries and vein were removed, Wharton’s jelly was finely dissected into small pieces. The pieces were individually placed on 100-mm2 tissue culture dishes with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Gibco) containing 10% fetal bovine serum (FBS; Gibco), and 1% P/S (Gibco) and incubated for 10–12 days at 37 °C with 5% CO2. The medium was subsequently exchanged every 2–3 days. The cultures were passaged when they reached 80–90% confluency after carefully removing the umbilical cord tissues. hUC-MSCs at passage 3 were cultured to 80–90% confluence in a T75 culture flask (about 10^6 cells). The complete medium was replaced with serum-free DMEM/F-12 medium (5 ml) to avoid peptides contamination from FBS. The collected serum-free medium was centrifuged for 10 min at 300 g at 4 °C to remove cell debris, and protease inhibitor (Roche, Basel, Switzerland) was added. Lastly, the mixture was snap-frozen in liquid nitrogen and stored at − 80 °C until used, as previously described [36].

2.3 Cell surface marker analysis

Using hUC-MSCs at passage 4, flow cytometry was conducted using a Gallios Flow Cytometer (Beckman Coulter, Fullerton, CA, USA) to assess the cell surface markers. The cells were washed with precooled PBS followed by staining with the following mouse antibodies: allophycocyanin (APC)-conjugated anti-CD29 (Miltenyi, Bergisch Gladbach, Germany), phycoerythrin (PE)-conjugated anti-CD73 (Miltenyi), fluorescein isothiocyanate (FITC)-conjugated anti-CD105 (Biolegend, San Diego, CA, USA), PE-conjugated anti-CD31 (eBioscience, San Diego, CA, USA), PE-conjugated anti-CD34 (eBioscience) and APC-conjugated anti-HLA-DR (Miltenyi). Kaluza 1.2 cytometry software (Beckman Coulter) was used for the hUC-MSC cell surface marker analysis.

2.4 Cell differentiation

The differentiation potential of hUC-MSC cultures, regarding chondrocyte and adipocyte lineages, was assessed using a StemPro® Chondrogenesis Differentiation Kit (Gibco) and Preadipocyte Differentiation Medium (ScienCell, San Diego, CA, USA) according to the manufacturers’ instructions. Briefly, at 70–80% confluency, hUC-MSCs were incubated in prewarmed complete differentiation medium with supplements. The differentiation medium was changed every 3 days. After 21 days under differentiation conditions, the cells were washed with 1x PBS (Gibco) and then fixed with 4% paraformaldehyde for 30 min at room temperature (RT). After rinsing with 1x PBS (Gibco), the cells were stained with Oil Red O (Sigma-Aldrich, Saint Louis, MO, USA, for adipocytes) and Alcian blue (Servicebio, Wuhan, China, for chondrocytes) according to the manufacturers’ guidelines.

2.5 Peptide extraction and purification

Before peptide extraction, the protein integrity of the CM samples was appraised. The collected CM samples were concentrated by centrifugation under vacuum (LaboGene, Allerød, Denmark), boiled in sodium dodecyl sulfate (SDS)-sample buffer at 95℃ for 10 min and then subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE). The SDS-PAGE gel was stained using a Pierce Silver Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Thereafter, the samples were filtered through an ultrafiltration tube (Amicon Ultra-15, Millipore, MA, USA) with a molecular weight cutoff (MWCO) of 10 kDa to acquire the filtered liquid containing the peptides. The protein concentration of the supernatant was also measured using the Bradford protein assay [37].

2.6 TMT labelling and LC-MS/MS analysis

The peptides from preterm and term hUC-MSC CM were reduced with 10 mM DL-dithiothreitol (DTT; Promega, WI, USA) for 1 h at 56 °C and alkylated with 55 mM iodoacetamide (Promega) for 1 h in the dark at RT. Thereafter, precooled acetone was added, and the peptides were precipitated over 3 h at -20 °C. After centrifuging for 20 min at 20000 g and 4℃, the precipitate was dissolved in 300 µl of the following buffer: 50% triethylamine borane (Sigma) and 0.1% SDS (Sigma). Next, the peptide solution was desalted using a Strata-X C18 column (Phenomenex, Torrance, CA, USA), dried and labeled with TMT reagent (TMT 6-plex Label Reagent; Thermo Fisher Scientific) for 1 h [38]. Next, the preterm and term samples were mixed at a 1:1 ratio on the basis of the total peptide amount. Analysis of labelled peptides was performed on a Q Exactive Orbitrap LC-MS/MS system (Thermo Fisher Scientific). Qualitative and relative quantitative analyses of the detected peptides were performed using the SWISSPROT_human database and Mascot software (version 2.3.01). Peptides with absolute fold change ≥ 1.5 and P value < 0.05 were considered differentially expressed.

2.7 Bioinformatics analysis

The molecular weight (MW) and isoelectric point (PI) of the identified peptides were calculated using the online tool PI/MW (http://web.expasy.org/compute.pi/) [39]. A Gene Ontology (GO) analysis (http://www.blast2go.com/b2ghome) was carried out to explore the possible cellular components, biological processes and molecular functions related to the precursor proteins [40]. Diseases and Regulator Effects networks analysis of the differentially expressed peptides and their precursors were performed using Ingenuity Pathway Analysis (IPA) software v7.1 (Ingenuity Systems, Mountain View, CA, USA) [39, 41]. The UniProt database (http://www.uniprot.org/) was used to detect the predominant subcellular locations of the precursors of the differentially expressed peptides. The Open Targets Platform database (www. targetvalidation.org/) was applied to study the diseases associated with the protein precursors [42].

2.8 Function analysis of differentially expressed peptides in vitro

We selected one peptide with high fold change and investigated the effects on human lung epithelial cells A549 (Meiyan, Shanghai, China) stimulated by hydrogen peroxide (H2O2; Kelong, Chengdu, Sichuan, China). The peptides were synthesized by GenScript (Nanjing, Jiangsu, China). The human lung epithelial cell lines A549 were cultured in Dulbecco’s Modified Eagle Medium (Gibco) with 10% FBS and 1% P/S. The A549 cells were exposed to 1 mM H2O2 with or without peptides (1 µM, 10 µM and 100 µM) for 24 h in serum-free DMEM with 1% P/S. then A549 cells were washed with PBS, and Total RNA was isolated using TRizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Reverse transcription reactions and real-time PCR were carried out to measure the expression levels of tumor necrosis factor α (TNFα) and interleukin-1β (IL 1β) mRNAs in A549 cells using SYBR Green Master Mix Kit (Roche). Primer sequences were: human TNFα: forward, 5’ CCTCTCTCTAATCAGCCCTCTG 3’, reverse, 5’ GAGGACCTGGGAGTAGATGAG 3’; human IL 1β: forward, 5’ AGCTACGAATCTCCGACCAC 3’, reverse, 5’ CGTTATCCCATGTGTCGAAGAA 3’; human β-actin: forward, 5’ AGCGAGCATCCCCCAAAGTT 3’, reverse, 5’ GGGCACGAAGGCTCATCATT 3’. To calculate fold change in the expression of these genes, ΔCt = Ct of individual genes - Ct of β-actin was first obtained. ΔΔCt = ΔCt of treated groups - ΔCt of control groups was then obtained. Fold change was calculated as 2−ΔΔCt, with control groups as 1.0 fold.

2.9 Statistical analysis

Student's t-test or one-way ANOVA was employed for statistical comparisons. The results of the bioinformatics analysis were visualized using GraphPad Prism 5/7 software. The statistical significances were calculated as P values, and P < 0.05 was considered statistically significant.

3. Results

3.1 Isolation and characterization of MSCs derived from human umbilical cords

The range of gestational ages (GA) was 31–32 weeks of gestation for the preterm umbilical cords, and 40–41 weeks for the term cords (Table 1). The hUC-MSCs (obtained by the tissue explants adherent method) had a typical fibroblast phenotype (Fig. 1A). Both preterm and term hUC-MSCs were positive for CD29, CD73 and CD105 staining and negative for CD31, CD34 and HLA-DR staining (Fig. 1B). Furthermore, these isolated cells had the potential to differentiate into adipocytes and chondrocytes (Fig. 1A). These results confirmed that MSCs from human umbilical cords were successfully isolated, without significant differences in morphology, expression of cell surface markers or differentiation capacities between the preterm and term groups.

3.2 Identification of differentially expressed peptides in hUC-MSC CM from preterm and term infants

We verified the protein integrity of the hUC-MSC CM by silver staining (Figure S1). The peptides from preterm and term hUC-MSC CM were directly analyzed by the TMT labeling method combined with LC-MS/MS. We identified a total of 3099 peptides in hUC-MSC CM from both groups. A total of 131 peptides were observed to be significantly differentially expressed (absolute fold change ≥ 1.5, P value < 0.05) in the hUC-MSC CM from the preterm group compared with the term group, comprising 37 up-regulated peptides (Fig. 2A) and 94 down-regulated peptides (Fig. 2B). The top 20 up-regulated and top 20 down-regulated peptides are shown with their precursor proteins in Figs. 2C and 2D. All the differentially expressed peptides are shown in Table 2.

3.3 Basic characteristics of the differentially expressed peptides in hUC-MSC CM from preterm and term infants

The MW and PI of the differentially expressed peptides were analyzed. The MW of most peptides ranged from 500 to 700 Da (Fig. 3A), and the PI ranged from 3 to 11 (Fig. 3B). We also investigated the distribution of the MW relative to the PI (Fig. 3C). Peptides are cleaved from their precursor proteins by specific enzymes [41], so we analyzed the cleavage sites at the N- and C-terminals of the identified peptides. Lysine (K) was the most common N-terminal amino acid (accounting for 13.7% of the peptides), while asparagine (N) was the most common C-terminal amino acid (accounting for 16.0% of the peptides) (Fig. 3D).

3.4 GO analysis and subcellular location analysis of the differentially expressed peptide precursors

Next, molecular functions, cellular components and biological processes of the corresponding precursor proteins were determined by GO analysis to predict the latent functions of the differentially expressed peptides. Binding and catalytic activity were the most highly enriched molecular functions (Fig. 4A). Cell part, organelle part and intrinsic component of membrane were the most highly enriched cellular components (Fig. 4B). Cellular process, biological regulation and cellular component organization were the most highly enriched biological processes (Fig. 4C). Furthermore, we categorized the subcellular locations of the precursor proteins of all 131 peptides in accordance with their annotations in the UniProt database. The analysis revealed that the nucleus (25%), plasma membrane (16%) and cytoskeleton (15%) were the predominant subcellular locations of the differentially expressed peptide precursors. About 10% of the precursors were types of proteins that are located in the extracellular region of hUC-MSCs (Fig. 4D).

3.5 Diseases and Regulator Effects networks associated with the differentially expressed peptide precursors

We further evaluated the diseases and Regulator Effects networks associated with the differentially expressed peptide precursors using IPA software. Disease and functional protein network analysis indicated that several precursor proteins were involved in developmental disorders and inflammatory responses (Figs. 5A and B). More precisely, precursor proteins including Alpha-2A adrenergic receptor (ADRA2A), Protein argonaute-2 (AGO2), Baculoviral IAP repeat-containing protein 6 (BIRC6), Kalirin (KALRN) and Histone-lysine N-methyltransferase 2C (KMT2C) were involved in developmental disorders, and KMT2C, Solute carrier family 2 (SLC2A4), Electrogenic sodium bicarbonate cotransporter 1 (SLC4A4), STIM1L (STIM1) and Rootletin (CROCC) were involved in inflammatory responses. All the putative precursor proteins associated with diseases are shown in Table S1. Furthermore, the Regulator Effects network analysis showed that some of the protein precursors participated in the networks of cellular development, embryonic development, organismal development and organismal injury and abnormalities (Figs. 5C and D). For example, AGO2, CROCC, DENN domain-containing protein 2A (DENND2A), Krueppel-like factor 14 (KLF14) and Lon protease homolog (LONP1) were involved in the networks of cellular development, embryonic development and organismal development. Additionally, Atrophin-1 (ATN1), Collagen alpha-1 (VIII) chain (COL8A1), Protein jagged-2 (JAG2), KMT2C and Mucin-19 (MUC19) were related to the network of organismal injury and abnormalities. All the precursor proteins involved in Regulator Effects networks are shown in Table S2.

3.6 Putative bioactive peptides associated with respiratory diseases

It is well known that peptides with biological functions have functions that are related to the functions of their precursor proteins, with domains playing key roles in the biological functions [43, 44]. The UniProt database was used to analyze the peptides and their precursors and the results showed that 25 peptides were located in the functional domains of their corresponding precursors (Table S3). The preceding results suggested that these precursors are mainly associated with inflammatory responses and abnormal organ development, which contribute to premature infant respiratory diseases [45]. Therefore, we focused on peptides and their precursors related to respiratory diseases. Using the Open Targets Platform database, we investigated whether these peptides might play potential roles in respiratory diseases. All told, 17 precursor proteins were found to be closely related to respiratory diseases (association score ≥ 0.5) (Table S4).

3.7 The effect of differentially expressed peptides in lung epithelial cells

Previous researches have reported that lung epithelial cells A549 can be stimulated by H2O2 to induce inflammatory response [46, 47]. Therefore we studied the effect of differentially expressed peptides on H2O2-treated A549 cells. According to the high fold change, we selected two peptides 508AAAAGPANVH517 derived from Homeobox protein SIX5 (SIX5, absolute fold change: 5.9) and 7118TGAKIKLVGT7127 derived from MUC19 (absolute fold change: 6.3) for future study. H2O2 (1 mM) treatment significantly increased the expression levels of TNFα and IL 1β mRNA in A549 cells. And the TNFα and IL 1β mRNA levels were significantly reduced in H2O2-treated A549 cells with peptide 7118TGAKIKLVGT7127 (MUC19) at 10 and 100 µM, but not at 1 µM (Figs. 6A). Meanwhile the TNFα and IL 1β mRNA levels were significantly reduced in H2O2-treated A549 cells with peptide 508AAAAGPANVH517 (SIX5) at 100 µM, but not at 1 µM or 10 µM (Figs. 6B).

4. Discussion

The MSC secretome and its therapeutic effects have been extensively demonstrated in preterm diseases, such as BPD [48] and NEC [19]. Previously, most researchers considered soluble factors and extracellular vesicles as the primary components of the secretome derived from MSCs [49–51]. However, the paracrine substances from MSCs are not limited to these biomolecules. Therefore, further studies are required to explore more types of components derived from MSCs and investigate their functions.

With the advance of detection technologies, mounting evidence has confirmed the differences in hUC-MSCs between preterm and term groups, which may help to identify the possible regulators or mechanisms underlying MSC function. To compare the global gene expression patterns in hUC-MSCs between these two groups, Iwatani et al. used microarray analysis and revealed that up-regulated WNT2B in preterm hUC-MSCs was involved in the control of hUC-MSC proliferation [52]. A very recent study comparing hUC-MSC transcriptomics and proteomics profiles from term and preterm groups showed that Frizzled-2 (FZD2) protein and mRNA expression levels were both higher in preterm hUC-MSCs [53]. Importantly, FZD2 is the receptor of Wnt5a/b and FZD2 mutations influence Wnt signaling, which mediates the epithelial to mesenchymal transition (EMT) during lung development [54–56]. In addition, by comparing the proteome of microvesicles collected from hUC-MSC CM between preterm and term groups, Bruschi et al. found that 173 proteins were significantly changed, 163 of which were increased in the preterm group [57]. However, there have been no comprehensive comparisons of hUC-MSC CM peptidomic profiles between preterm and term infants. In the present study, we found that 131 peptides derived from 106 precursor proteins were differentially expressed in the preterm hUC-MSC CM compared with the term group by TMT labeling quantification (Fig. 2). Our study provides hUC-MSC CM polypeptide profiles for preterm and term infants. Secreted peptides have been shown to have important biological functions. Neuropeptide Y, a 36-amino acid peptide secreted by the hypothalamus, was found to play key roles in neurodegenerative diseases including modulation of neurogenesis, food intake and thermogenesis [58–60]. Mao et al. showed that peptides derived from human beta-defensins are secreted by viable human cryopreserved amniotic membrane and exhibited direct antimicrobial effects against P. aeruginosa [61]. Further studies are needed to understand and explore the functions of secreted peptides from hUC-MSCs.

Subcellular location analysis of precursor proteins can help to better understand the source and potential functions of peptides. As shown in Fig. 5D, most precursor proteins were annotated as being part of organelles or membranes (56%). Notably, a small fraction of peptides were annotated as being derived from proteins located in the extracellular region, termed secreted proteins from hUC-MSCs. Classically, a large proportion of peptides (such as peptide neurotransmitters) are generated by the proteolysis of macromolecular proteins followed by release into the space outside of cells [62]. However, some other peptides containing one or more cleavage sites do not derive from endosomal processing [63]. These peptides are the N- or C-terminal peptides of their precursor proteins, rather than internal fragments [64]. In addition, the identification in our study of several peptides arising from secreted proteins raised the possibility that some bioactive peptides may be produced by enzymatic hydrolysis of extracellular proteins. These observations provide us with more methods to evaluate the potential functions of differentially expressed peptides in our study; additional studies are needed to ascertain whether these peptides are actually secreted from hUC-MSCs and have biological activities.

From the results of IPA analysis, we found that a precursor protein named KMT2C was involved in networks related to both developmental disorders and inflammatory responses. In addition, KMT2C was clearly associated with respiratory diseases according to the Open Targets Platform database. KMT2C, a member of the KMT2 family, is a putative tumor suppressor in several epithelial cells including pulmonary epithelial cells [65, 66]. KMT2C knockdown has been shown to promote EMT, while forced expression of KMT2C decreased the expression of EMT-related proteins [67]. EMT is considered an important mechanism underlying neonatal respiratory disease [45, 68, 69]. Thus, peptides derived from the KMT2C protein (746EGCVK750 and 3890QQNNLSNP3897) may influence neonatal respiratory diseases.

Additionally, among the differentially secreted peptides in hUC-MSC CM, we found that a peptide derived from KLF14 (961DDFMSSQN968) was significantly up-regulated in the preterm group. In agreement with this, KLF14 expression was previously found to be up-regulated in preterm hUC-MSCs [52]. This consistent expression pattern suggests a similar role of the peptide as for KLF14. The upstream Regulator Effects networks analysis of peptide precursors also showed that KLF14 participates in embryonic and organismal development. KLF14 is a zinc finger DNA-binding protein belonging to the Specificity protein/Kruppel-like factor (SP/KLF) family [70]. Both the KLF14 mRNA and protein levels were reduced in lung tissues from lipopolysaccharide-treated mice and KLF14 overexpression inhibited inflammation in human coronary artery endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) [71]. Taken together, it appears that KLF14-derived peptide may contribute to inflammation inhibition in neonatal respiratory diseases. On the whole, these observations raised the possibility that the peptides secreted by hUC-MSCs may have beneficial effects in neonatal respiratory diseases, which are worthy of in-depth functional studies.

As the functions of secretory peptides derived from hUC-MSCs were unclear, we assessed whether the peptides were located in the functional domains of their precursor proteins to analyze their functionality. Using the UniProt database, we discovered that 25 peptides were situated in the functional domains of their corresponding precursors. Peptides derived from Titin (8990ESDSG8994, 26941EGNKDD26946, 6684SSRLECKI6691 and 19103VVHAGGVIRIIAYV19116) and one peptide derived from striated muscle preferentially expressed protein kinase (2669SCTVAVARVPGKLAPPEVPQ2688) were located in Ig-like domains. The Ig-like domain was initially characterized as a structure composed of two sheets of antiparallel β strands [72]. Okano et al. reported that the Ig-like domain contributed to the maintenance of the structure, activity and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase [73]. Interestingly, the Ig-like domain made endoglucanase Cel9A from Alicyclobacillus acidocaldarius dependent on calcium [74]. Additionally, Ig-like domains have been shown to play various roles in functions [75, 76] and binding [43, 77]. We also found that two peptides derived from MUC19 (961DDFMSSQN968 and 1382QNGIIVI1388) were located in the type D von Willebrand factor (VWFD) domain. This domain plays a substantial role in reducing soluble VWF binding to platelet GpIba and regulates platelet activation and adhesion [78, 79]. It also participates in fertilization, as it binds to sperm proteases [80]. Like the Ig-like domain, the VWFD domain is partially responsible for biological processes and functions. The above motif analysis may provide a new perspective for clarifying the possible roles of these newly identified peptides.

Then more investigations were carried out to investigate the effect of differentially expressed peptides in vitro. Premature respiratory diseases including BPD are mostly related to lung development, oxidative stress and inflammatory response[81, 82]. H2O2 is a strong two-electron oxidant which contributes to cell damage or death [83, 84]. And Wang Wei et al. showed that H2O2 induced inflammatory injury in human lung epithelial cell line A549 [85]. Airway epithelial cells are the first defense cells against environmental stimuli [86], consequently we chose human lung epithelial cells A549 to explore the effects of differentially expressed peptides in vitro. We found that the TNFα and IL 1β mRNA levels increased in vitro H2O2 stimulation of A549 cells as shown in Fig. 6. Our results showed that 7118TGAKIKLVGT7127 derived from MUC19 reduced the levels of TNFα and IL 1β mRNA in H2O2-treated A549 cells and was down-regulated in hUC-MSC CM from preterm group. MUC19 as a secreted mucin, is released to the extracellular medium and has been identified in respiratory, digestive, and reproductive tracts[87]. It has been reported that MUC19 was differentially regulated after exposure to inflammatory cytokines[88]. And one recent study found that MUC19 peptides may enhance vaginal mucous immunity against infections[89]. These results indicated that 7118TGAKIKLVGT7127 derived from MUC19 may play a role in reducing inflammatory response reduced by H2O2 in human lung epithelial cell which needs more future investigations. In addition, the other peptide 508AAAAGPANVH517 derived from SIX5 also reduced the levels of TNFα and IL 1β mRNA in H2O2-treated A549 cells. SIX5, previously known as myotonic dystrophy associated homeodomain protein-DMAHP, is a member of the SIX (sine oculis homepbox (Drosophila) homologue) family of transcription factors [90]. It has been acknowledged that SIX5 was correlated with eye development [91], Myotonic dystrophy [92], Branchio-oto-renal syndrome [93], which are related to embryonic and organismal development. And the peptide 508AAAAGPANVH517 derived from SIX5 was up-regulated in hUC-MSC CM from preterm group. And Chaubey et al. found that MSC-derived exosomes from preterm infants repaired the lung injury and improved BPD-associated brain injury[36]. So we put forward one hypothesis that the hUC-MSCs from preterm infants may secrete protective substances under stress such as peptides. These findings will be important for future investigations in the roles of differentially expressed peptides and their possible mechanism in premature respiratory diseases.

5. Conclusion

As far as we know, no large-scale quantitative peptidomic analysis has been carried out on the secretory components of hUC-MSCs. Our study identified the differentially expressed peptides secreted by preterm and term hUC-MSCs using TMT-based LC-MS/MS technology. Furthermore, bioinformatics analysis of precursors predicted the possible functions of peptides that may be useful in the treatment of premature respiratory diseases in connection with inflammatory responses and developmental disorders. And we also preliminarily investigated the effect the peptide 508AAAAGPANVH517 derived from SIX5 on human lung epithelial cells. This study expands our knowledge of the hUC-MSC secretome and may provide insights into new therapy for premature respiratory diseases. There are some limitations in our research. First, we used P-value to screen differentially expressed peptides between term and preterm groups. Although the false discovery rate (FDR) can detect differentially expressed peptides more accurately than p value, we would get very few peptides by FDR. Because we found that some studies also applied the P value to the analysis of peptides [94–96], we also used the p value in the study. Secondly, we preliminarily explored the functions of differentially expressed peptides on H2O2-treated A549 cells, and the cellular mechanisms and target molecules of these peptides are unknown, which need more detailed studies in vivo and in vitro.

Abbreviations

UC-MSC

umbilical cord mesenchymal stem cell;

TMT

tandem mass tag;

CM

conditioned medium;

GO

Gene Ontology;

IPA

Ingenuity Pathway Analysis;

PVL

periventricular leukomalacia;

BPD

bronchopulmonary dysplasia;

NEC

necrotizing enterocolitis;

ROP

retinopathy of prematurity;

Ang-1

Angiopoietin-1;

HO-1

Heme oxygenase-1;

EPO

Erythropoietin;

GLP-1

Glucagon-like peptide-1;

CGRP

calcitonin gene-related peptide;

AEC II

alveolar epithelium type II cells;

LC-MS/MS

liquid chromatography-tandem mass spectrometry;

DMEM/F-12

Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12;

RT

room temperature;

SDS

sodium dodecyl sulfate;

MWCO

molecular weight cutoff;

MW

molecular weightand;

PI

isoelectric point;

H2O2

hydrogen peroxide;

TNFα

tumor necrosis factor α;

IL 1β

interleukin 1β;

GA

gestational ages;

ADRA2A

Alpha-2A adrenergic receptor;

AGO2

Protein argonaute-2;

BIRC6

Baculoviral IAP repeat-containing protein 6;

KALRN

Kalirin;

KMT2C

Histone-lysine N-methyltransferase 2C;

SLC2A4

Solute carrier family 2;

SLC4A4

Electrogenic sodium bicarbonate cotransporter 1;

CROCC

Rootletin;

DENND2A

DENN domain-containing protein 2A;

KLF14

Krueppel-like factor 14;

LONP1

Lon protease homolog;

ATN1

Atrophin-1;

COL8A1

Collagen alpha-1 (VIII) chain;

JAG2

Protein jagged-2;

MUC19

Mucin-19;

MPDZ

Multiple PDZ domain protein;

SIX5

Homeobox protein SIX5;

FZD2

Frizzled-2;

EMT

epithelial to mesenchymal transition;

HCAECs

human coronary artery endothelial cells;

HUVECs

human umbilical vein endothelial cells;

VWFD

type D von Willebrand factor.

Declarations

Ethics approval and consent to participate

This study has been approved by the Ethics Committee of Changzhou Maternal and Child Health Care Hospital (approval number: 2019126). Written consent to participate was obtained from the parents of the patients.

Consent for publication

 Written informed consent for publication of their clinical details was obtained from the parents of the patients.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

Contract grant sponsors and numbers: Jiangsu Provincial Women and Children Health Research Project (grant no. F201816 and F201744), Changzhou Science and Technology Planning Project (grant no. CE20165050), National Natural Science Foundation of China (grant no. 81600687 81701491 and 81501257), Nanjing Medical Science and Technique Development Foundation (grant no. QRX17160 and YKK18155), Jiangsu Provincial Medical Youth Talent (grant no. QNRC2016111), Six Talent Peaks Project of Jiangsu Province (grant no. YY-112), Jiangsu Province Natural Science Foundation (grant no. BK20170152) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (grant no. JX22013535).

Author Contributions

YuW and LZ performed the experiments, interpreted the results of the experiments and drafted the manuscript. YunW and RPZ prepared the figures. YanW and YC analyzed the data. WL and CBJ participated in the discussion. HYW and LHY conceived and designed the experiments, provided funding to regents. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by Jiangsu Provincial Women and Children Health Research Project (grant no. F201816 and F201744), Changzhou Science and Technology Planning Project (grant no. CE20165050), National Natural Science Foundation of China (grant no. 81600687 81701491 and 81501257), Nanjing Medical Science and Technique Development Foundation (grant no. QRX17160 and YKK18155), Jiangsu Provincial Medical Youth Talent (grant no. QNRC2016111), Six Talent Peaks Project of Jiangsu Province (grant no. YY-112), Jiangsu Province Natural Science Foundation (grant no. BK20170152) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (grant no. JX22013535). The TMT method followed by mass spectrometry analysis was supported by the Analysis and Testing Center of Nanjing Medical University.

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Tables



Table 2 Differentially expressed peptides in hUC-MSC conditioned medium from preterm and term infants

Accession

Gene

Protein

Petide

MW(kD)

Fold change

-10lgP

Up-regulated peptides

Q6PEZ8-3

PODNL1

Podocan-like protein 1

PSLERLHLQNNLISKVPR

5.94 

∞

6.96 

A0A0A0MTS7

TTN

Titin

ESDSG

2.92 

13.62 

9.66 

Q9BYJ1-2

ALOXE3

Hydroperoxide isomerase ALOXE3

LNGRQQY

0.54 

7.95 

10.08 

Q15772-1

SPEG

Striated muscle preferentially expressed protein kinase

SCTVAVARVPGKLAPPEVPQ

0.76 

5.96 

10.05 

Q8N196

SIX5

Homeobox protein SIX5

AAAAGPANVH

1.00 

5.93 

11.82 

P49815-3

TSC2

Tuberin

PAGPAVRL

0.57 

4.52 

10.67 

A0A0A6YYA3

CDHR1

Cadherin-related family member 1

RVLRKRPSPAPRTIRIE

0.69 

3.82 

9.34 

Q7Z5P9-2

MUC19

Mucin-19

DDFMSSQN

2.05 

3.46 

5.01 

P54259

ATN1

Atrophin-1

GPARPYHP

0.67 

3.28 

11.27 

A0A140TA73

SNTB2

Beta-2-syntrophin

NGLPNGGGAGDS

0.88 

2.98 

7.03 

Q9UQD0-2

SCN8A

Sodium channel protein type 8 subunit alpha

EAGID

1.81 

2.67 

16.18 

Q5TZA2-2

CROCC

Rootletin

RLLKGEASLEV

0.68 

2.67 

9.78 

E7EMZ9

TACC2

Transforming acidic coiled-coil-containing protein 2

RMSESPTPC

1.29 

2.57 

8.92 

A0A075B756

KLF14

Krueppel-like factor 14

TKHARRHP

0.94 

2.52 

17.50 

A0A0A0MTS7

TTN

Titin

LEDGG

3.06 

2.50 

12.31 

O15027-2

SEC16A

Protein transport protein Sec16A

KSILTQ

2.31 

2.49 

13.78 

Q9NRA0-3

SPHK2

Sphingosine kinase 2

EWDGIVTVSGDGLLHEVLN

0.56 

2.46 

10.14 

Q8WXG9

ADGRV1

Adhesion G-protein coupled receptor V1

EAGLD

2.02 

2.23 

19.57 

Q9Y4D8

HECTD4

Probable E3 ubiquitin-protein ligase HECTD4

KLAKLQRIARQAVAALCALGG

1.02 

2.21 

6.58 

A0A087WVF8

PDE4DIP

Myomegalin

QSMMAV

1.01 

2.18 

21.26 

Q01167

FOXK2

Forkhead box protein K2

QTVHVVH

0.67 

2.18 

12.99 

Q5IJ48

CRB2

Protein crumbs homolog 2

LLEVAVPAACACLLLLLLGLLSGILAARK

0.78 

2.18 

5.08 

Q9ULE3

DENND2A

DENN domain-containing protein 2A

FLHKK

1.70 

2.17 

21.46 

P53420

COL4A4

Collagen alpha-4(IV) chain

PGEPGLVGPPGQPGRPG

0.84 

2.14 

7.51 

Q7Z5P9-2

MUC19

Mucin-19

FLGGS

2.11 

2.11 

13.79 

P01833

PIGR

Polymeric immunoglobulin receptor

QADGSRASVD

0.49 

2.10 

5.11 

Q09666

AHNAK

Neuroblast differentiation-associated protein AHNAK

KLKGDI

1.57 

2.08 

15.35 

P36776

LONP1

Lon protease homolog, mitochondrial

KHKPR

0.99 

2.07 

8.69 

A0A0A0MTS7

TTN

Titin

VPEAPKEVVPEKKVPVTPPKK

2.94 

1.98 

10.42 

Q8NEZ4

KMT2C

Histone-lysine N-methyltransferase 2C

QQNNLSNP

1.03 

1.96 

10.44 

A0A0A0MTS7

TTN

Titin

SPPSP

2.94 

1.93 

11.70 

Q7Z5P9-2

MUC19

Mucin-19

AGTSI

2.24 

1.89 

23.15 

O75592-2

MYCBP2

E3 ubiquitin-protein ligase MYCBP2

QLLYR

2.05 

1.79 

11.10 

Q9H6K5-2

PRR36

Proline-rich protein 36

PPSLQTLPSPPATPPSQVPPTQ

0.81 

1.78 

9.94 

P08913

ADRA2A

Alpha-2A adrenergic receptor

ISAVISFPPLISIEKKGGGG

0.93 

1.71 

9.59 

Q9UJ55

MAGEL2

MAGE-like protein 2

PPPIRPGP

1.10 

1.65 

15.90 

Q4V328-4

GRIPAP1

GRIP1-associated protein 1

LCSQMEQLE

0.63 

1.60 

6.91 

Down-regulated peptides

 

 

 

 

 

 

O14526-3

FCHO1

F-BAR domain only protein 1

AGIVRVF

0.53 

-1.56 

9.90 

Q7Z5P9-2

MUC19

Mucin-19

KTLAAGS

2.15 

-1.57 

14.06 

Q99814

EPAS1

Endothelial PAS domain-containing protein 1

TPLSSMGGRS

1.00 

-1.58 

18.34 

Q6ZNL6

FGD5

FYVE, RhoGEF and PH domain-containing protein 5

EDHAQ

0.77 

-1.58 

13.24 

Q9BW04-2

SARG

Specifically androgen-regulated gene protein

LTTPKPRKLPPN

0.61 

-1.60 

5.40 

O15027-2

SEC16A

Protein transport protein Sec16A

QACAASGS

2.41 

-1.61 

17.59 

S4R393

ZSWIM8

Zinc finger SWIM domain-containing protein 8

QTHKPQT

0.99 

-1.64 

13.03 

Q2VWA4

SKOR2

SKI family transcriptional corepressor 2

GGSGGDCSAG

0.50 

-1.65 

8.13 

Q9Y6V0-6

PCLO

Protein piccolo

QQPGPAKPPP

1.00 

-1.69 

6.21 

P28329

CHAT

Choline O-acetyltransferase

GLPKLPVPPLQQ

0.66 

-1.70 

5.12 

Q8WXH0

SYNE2

Nesprin-2

KIYKKFLKKAQDLTSLLKEL

2.04 

-1.71 

5.55 

P13611-5

VCAN

Versican core protein

QPEFSS

1.97 

-1.74 

29.72 

Q8TE85

GRHL3

Grainyhead-like protein 3 homolog

LFIPNVHFSSLQRSG

0.54 

-1.78 

9.85 

A0A1B0GUF7

IQCM

IQ domain-containing protein M

KTFKT

0.88 

-1.79 

14.67 

A0A087WXW9

COL5A1

Collagen alpha-1(V) chain

PPGEV

2.70 

-1.83 

8.75 

A0A0A0MTS7

TTN

Titin

KACDPVF

2.92 

-1.87 

8.46 

A0A0A0MTS7

TTN

Titin

IVASDVTKRLIKANLLANN

2.78 

-1.87 

5.62 

H0Y5I7

SFI1

Protein SFI1 homolog

QQLAARRQEQRATVRALW

0.82 

-1.88 

6.99 

A6NMZ7

COL6A6

Collagen alpha-6(VI) chain

RRAIN

0.91 

-1.89 

13.43 

Q8NEZ4

KMT2C

Histone-lysine N-methyltransferase 2C

EGCVK

1.08 

-1.89 

12.10 

A0A087WXW9

COL5A1

Collagen alpha-1(V) chain

GPRGITGKPGPK

2.70 

-1.90 

10.79 

Q9Y6W6

DUSP10

Dual specificity protein phosphatase 10

DNQAQT

1.21 

-1.91 

9.85 

A0A0J9YXV3

N/A

Uncharacterized protein

KIGLGY

0.94 

-1.91 

14.05 

A0A0A0MTS7

TTN

Titin

EGNKDD

3.08 

-1.91 

12.34 

Q92616

GCN1

eIF-2-alpha kinase activator GCN1

ILDVASLEVLN

0.66 

-1.92 

5.74 

Q9NR09

BIRC6

Baculoviral IAP repeat-containing protein 6

DNESCTN

1.47 

-1.95 

6.90 

Q8TEP8

CEP192

Centrosomal protein of 192 kDa

LLSTTK

1.70 

-1.96 

17.83 

A0A0A0MTS7

TTN

Titin

DPPGKPVPLN

3.22 

-2.08 

14.35 

Q8TE73

DNAH5

Dynein heavy chain 5, axonemal

QRVKSKIPAAIEQLIVPHLAKVDEALQPGLAAL

1.84 

-2.10 

7.15 

P10827-4

THRA

Thyroid hormone receptor alpha

LHARAV

0.51 

-2.11 

9.87 

Q9P2D3

HEATR5B

HEAT repeat-containing protein 5B

HAKGK

0.83 

-2.12 

12.50 

O60423-3

ATP8B3

Phospholipid-transporting ATPase IK

YGLVI

0.98 

-2.13 

11.77 

P06401-2

PGR

Progesterone receptor

GPLLKGKPRALGGAAAGGG

0.77 

-2.14 

5.47 

A0A140T8Y3

TNXB

Tenascin-X

HGRGRCEEGRCLCDPGYTGPTCATRMCPADCRGRGRCVQGVCLCHVGYGGEDCGQ

1.71 

-2.14 

5.92 

P27658

COL8A1

Collagen alpha-1(VIII) chain

GIDGVKPPHAYGAKKGKN

0.65 

-2.14 

6.82 

Q9C093

SPEF2

Sperm flagellar protein 2

ESLCEKVKEILTTEIAKKKN

0.69 

-2.14 

7.26 

Q8NAC3-3

IL17RC

Interleukin-17 receptor C

AAALSLILLLKKDHAKGWLRLLKQ

0.48 

-2.15 

5.00 

Q92771

DDX12P

Putative ATP-dependent RNA helicase DDX12

KGGLLGRLAARKKIFQEPK

0.67 

-2.16 

6.09 

Q6PJG9

LRFN4

Leucine-rich repeat and fibronectin type-III domain-containing protein 4

VAVGGVLVAALLVFTVALLVRGRGAGNGRL

0.69 

-2.17 

5.32 

Q9HD67

MYO10

Unconventional myosin-X

KTSCVE

0.82 

-2.23 

14.68 

Q96DN2

VWCE

von Willebrand factor C and EGF domain-containing protein

RPVLHLLQLLLRTNLMKTQTL

0.50 

-2.26 

9.34 

Q96QD8

SLC38A2

Sodium-coupled neutral amino acid transporter 2

VFNLSNAIVGSGILGLS

0.60 

-2.31 

12.68 

A0A0C4DGG6

NPC1L1

NPC1-like intracellular cholesterol transporter 1

VFAVVTILLVGFRVAPARDKSKMVDPKK

0.68 

-2.32 

12.53 

Q9UQD0-2

SCN8A

Sodium channel protein type 8 subunit alpha

VSLVSLIAN

1.71 

-2.33 

12.11 

Q9UPA5

BSN

Protein bassoon

KGGPRPR

2.05 

-2.34 

10.76 

Q9UKV8

AGO2

Protein argonaute-2

KLQAN

1.17 

-2.36 

13.86 

Q9UQD0-2

SCN8A

Sodium channel protein type 8 subunit alpha

YLALL

1.72 

-2.36 

14.90 

I6L894

ANK2

Ankyrin-2

KHKLNVP

0.89 

-2.38 

11.67 

O95996

APC2

Adenomatous polyposis coli protein 2

PAAEAATKKPLPPLRH

0.78 

-2.39 

5.35 

P36776

LONP1

Lon protease homolog, mitochondrial

TIAAKRAGVT

0.99 

-2.46 

12.42 

H7BXZ5

KALRN

Kalirin

VKKCIHKATRKDVAVKFVSKKMKKKEQA

0.91 

-2.46 

9.58 

Q8NF91-4

SYNE1

Nesprin-1

QKAVDHRKAIILSIN

1.02 

-2.56 

8.45 

J3KQC6

TMPRSS13

Transmembrane protease serine 13

LPLIGCVLLLIALVVSLIILFQFW

0.45 

-2.60 

5.00 

Q8TE73

DNAH5

Dynein heavy chain 5, axonemal

AQTKRLVGDVLLATAFLSYSGP

1.82 

-2.62 

5.48 

I3L2R4

SLC2A4

Solute carrier family 2 (Facilitated glucose transporter), member 4, isoform CRA_b

IGAGVVNTVFTLVSVLLVERAGRRTLHLLGLA

0.62 

-2.63 

7.43 

P42167

TMPO

Lamina-associated polypeptide 2, isoforms beta/gamma

KSEKTKKGRSIPVWIKILLFVVVAV

0.49 

-2.66 

6.27 

Q9BW11-4

MXD3

Max dimerization protein 3

GPIHRRK

0.50 

-2.66 

7.43 

E7EPG1

MMRN1

Multimerin-1

LPDIQLLQKGLTEFV

0.66 

-2.70 

6.46 

G5EA42

TMOD2

Tropomodulin 2 (Neuronal), isoform CRA_a

HVKKF

0.67 

-2.76 

12.93 

A0A0A0MTS7

TTN

Titin

SSRLECKI

2.84 

-2.77 

7.13 

Q7Z5P9-2

MUC19

Mucin-19

QNGIIVI

2.18 

-2.78 

18.23 

Q5H8A4

PIGG

GPI ethanolamine phosphate transferase 2

WLAAGGVMVLASALLCVIVSVLTNVLVGGN

1.18 

-2.80 

9.72 

Q9NR09

BIRC6

Baculoviral IAP repeat-containing protein 6

TRIGLKLIDILLRNCAAS

1.36 

-2.82 

5.14 

A0A0A0MTS7

TTN

Titin

VVHAGGVIRIIAYV

2.83 

-2.89 

5.92 

Q8NG04

SLC26A10

Solute carrier family 26 member 10

EPVVKALTSGAALHVLLSQLPSLLGLSL

0.59 

-2.91 

11.40 

J3KNF3

TET3

Methylcytosine dioxygenase TET3

GPEGCSA

1.65 

-2.94 

15.44 

Q92835-2

INPP5D

Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1

PLPVKSPA

1.01 

-2.97 

22.11 

Q9Y6R1

SLC4A4

Electrogenic sodium bicarbonate cotransporter 1

HHTIYIGVHVPKSYR

0.78 

-2.99 

8.24 

Q99707

MTR

Methionine synthase

KSARVMKKAVG

1.66 

-3.12 

21.33 

Q8WXG9

ADGRV1

Adhesion G-protein coupled receptor V1

RFLQSIYLVPEEDHILIIPVVRGKDN

1.97 

-3.13 

8.73 

P43243-2

MATR3

Matrin-3

HLILN

0.67 

-3.21 

8.71 

O75970-3

MPDZ

Multiple PDZ domain protein

FISLLKT

0.58 

-3.22 

11.08 

Q96N23-2

CFAP54

Cilia- and flagella-associated protein 54

HLKKPKIKISGSPLTLKPPLRRSSSVKET

0.62 

-3.25 

6.96 

F8WBW8

BAHCC1

BAH and coiled-coil domain-containing protein 1

RSVKAKVGTTL

1.16 

-3.27 

15.77 

F8VZY0

MYBPC1

Myosin-binding protein C, slow-type

TDAKIFVRVKAVNAAGAS

0.76 

-3.39 

9.25 

G0XQ39

STIM1

STIM1L

GVHPGSLVEKLPDSPALAKKALLALNHGL

0.59 

-3.56 

12.45 

P0CJ78

ZNF865

Zinc finger protein 865

MEANPAGSGAGGGGSSGIGGEDGVHFQSYPFDFLEFLNHQRFEPMELYGEHAKAVAA

0.86 

-3.84 

11.26 

Q7LBC6-2

KDM3B

Lysine-specific demethylase 3B

VKSKASLPN

0.69 

-3.87 

7.94 

Q9UI33-3

SCN11A

Sodium channel protein type 11 subunit alpha

IGAIPAILNV

0.83 

-4.13 

15.16 

Q5XPI4

RNF123

E3 ubiquitin-protein ligase RNF123

HYLRLTIAI

0.67 

-4.16 

8.06 

O15027-2

SEC16A

Protein transport protein Sec16A

AGSLCQALLPGPSNEAAGDVWGDTASTGVPDASGSQYE

2.30 

-4.48 

5.51 

B7ZLJ5

MPIG6B

C6orf25 protein

YPQLLIPLLGAGLVLGLGALG

0.48 

-4.66 

5.03 

Q86WI1

PKHD1L1

Fibrocystin-L

LFVGR

1.16 

-4.97 

17.10 

Q7Z3U7

MON2

Protein MON2 homolog

KPPQYGQLETKHIAN

1.01 

-5.28 

21.73 

F1T0I1

SEC16A

Protein transport protein sec16

RRRAN

2.30 

-5.79 

11.96 

A0A087WXW9

COL5A1

Collagen alpha-1(V) chain

HPGLI

2.50 

-5.82 

5.85 

Q9H3S7

PTPN23

Tyrosine-protein phosphatase non-receptor type 23

KLELLRQN

0.65 

-6.11 

5.79 

Q7Z5P9-2

MUC19

Mucin-19

TGAKIKLVGT

2.11 

-6.37 

6.06 

P52746

ZNF142

Zinc finger protein 142

TGLKP

4.94 

-∞

13.46 

P43088-7

PTGFR

Prostaglandin F2-alpha receptor

QKSKASFLL

3.34 

-∞

9.46 

A0A1C7CYW7

TTC34

Tetratricopeptide repeat protein 34

TGGQRLLAAL

3.68 

-∞

12.39 

Q9Y219

JAG2

Protein jagged-2

CGSDAGPGMPGTAASGVCGPHGRCVSQPGGN

3.52 

-∞

10.83 

E7ERG8

LRP1B

Low-density lipoprotein receptor-related protein 1B

QVDQFSCGNGRCIPRAWLCDREDDCGDQTDEMASCEFPTCEPLT

3.26 

-∞

5.58 

O75129-2

ASTN2

Astrotactin-2

TCHLC

5.80 

-∞

12.93 

 

Additional Files

Figure S1 Identification of protein integrity of hUC-MSC CM from preterm and term infants by silver staining. The protein integrity of hUC-MSC CM was visualized by SDS-PAGE and silver staining (n=3 per group, P1-3 represent preterm infants and T1-3 term represent term infants).

Table S1 Putative precursor proteins associated with diseases.

Table S2 Precursor proteins involved in networks.

Table S3 Differentially peptides located in functional domain based on Uniprot database.

Table S4 Protein precursors and identified peptides related to respiratory system diseases.