Protein difference between Pseudoglandular and Canalicular in the human fetal lung

An overview of the experimental measure had shown in Fig. 1A. We used Label Free technology to identified 6,943 proteins (5,929 quantitative proteins) for protein expression to Pseudoglandular and Canalicular in LC–MS/MS. Compared with the fetal lung of 17 weeks, there were 2,645 DEPs (different expression proteins) (Fig. 1B), including 1,402 proteins up-regulated (≥ 1.5-fold) and 1243 down-regulated (≤ 0.67-fold) in the fetal lung of 12 weeks. For protein crotonylation, we identified 15,427 crotonylation sites in 3,802 proteins (6,960 quantitative crotonylation sites in 2,217 proteins). Among then, 2,196 crotonylation sites were identified in 1,156 DCPs(different crotonylation proteins) between the fetal lung of 17 weeks and 12 weeks (Fig. 1C). One thousand three hundred fifty-nine crotonylation sites were up-regulated (≥ 1.5-fold) in 793 proteins, and 837 crotonylation sites were down-regulated (≤ 0.67-fold) in 586 proteins. The hierarchical clustering analysis of DEPs had shown in Fig. 1D. We overlapped to 514 proteins in all DEPs and DCPs (Fig. 1E). We analyzed the motifs of lysine crotonylation through Motif analysis, and there was no obvious amino acid enrichment in Top5 (Fig. 1F).

Fig. 1

Numerous Proteins Differentially Expressed in the Lung of Fetal Were Identified. A The schematic flow to study the proteome and crotonylome of fetal lung. B, C DEPs and DCPs between 12 and 17 weeks of fetal lung, respectively. D Hierarchical clustering analysis of DEPs. E Venn diagram between DEPs and DCPs. F Significantly enriched crotonylation motifs of top 5

Differentially quantified proteins to subcellular, KOG, and functional category characterization

To clarify the functions of these DEPs and DCPs, we analyzed the DEPs and DCPs using subcellular location (Fig. 2A, B). We found the top three: most DEPs are nucleus (n = 912, 34.49%), cytoplasm (n = 734, 27.76%), and mitochondria (n = 311, 11.76%); most DCPs are cytoplasm (n = 469, 40.57%), nucleus (n = 335, 28.98%), and mitochondria (n = 107, 9.26%). We also investigated the KOG categories of the DEPs and DCPs (Fig. 2C, D). We found the top three: most DEPs are [K] Transcription (n = 303), [T] Signal transduction mechanisms (n = 269), and [O] Posttranslational modification, protein turnover, chaperones (n = 217); most DCPs are [Z] Cytoskeleton (n = 334), [O] Posttranslational modification, protein turnover, chaperones (n = 234), and [J] Translation, ribosomal structure and biogenesis (n = 192).

Fig. 2

Subcellular, KOG, and Functional Category Characterization of Differentially Quantified Proteins. A, B Subcellular location of DEPs and DCPs, respectively. C, D KOG categories of DEPs and DCPs, respectively. E, F Functional category DEPs and DCPs in GO terms.

We adopted GO analysis tool to analyze DEPs and DCPs for functional category distribution (Fig. 2E, F). Three categories of GO of DEPs displayed: cellular component (92%), molecular function (81%), biological process (91%), and 24 biological groups; DCPs: functions were established for the DEPs and further divided into three classifications: cellular component (96%), molecular function (87%), biological process (94%), and 25 biological groups. Among them, most biological process of DEPs and DCPs: the cellular process (2,185 and 1,014, respectively), the biological regulation (1,726 and 823, respectively), and the metabolic process (1584 and 694, respectively). At the same, most cellular component of DEPs and DCPs: the cell (2,401 and 1,098, respectively) and the organelle (2,032 and 972, respectively). Most molecular function of DEPs and DCPs: the binding (1,667 and859, respectively) and the catalytic activity (1000 and 376, respectively).

Functional enrichment analysis of differentially quantified proteins

For the pathways analysis of GO in DEPs, we had been enriched: cellular component (n = 8), molecular function (n = 8), and biological process (n = 14) (Fig. 3A). The enrichment of biological process shows that protein targeting has regulated significantly, molecular function shows that histone binding has regulated significantly, and the cellular components show significant regulation in ribosomes. For the pathways analysis of GO in DCPs, we had been enriched: cellular component (n = 8), molecular function (n = 8), and biological process (n = 14) (Fig. 3B). The enrichment of the biological process shows that protein targeting has regulated significantly, the molecular function shows that the chromatin binding has regulated significantly, and the cellular components show that the vesicle has regulated significantly.

Fig. 3

Functional Enrichment Analysis of Differentially Quantified Proteins. A, B GO enrichment analysis of DEPs and DCPs, respectively. C, D Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of DEPs and DCPs, respectively

In KEGG pathway of DEPs, the significant ones were in Carbon metabolism, Starch and sucrose metabolism, Nitrogen metabolism, Tyrosine metabolism, DNA replication, and so on (Fig. 3C). Regarding crotonylation, the significantones were in Carbon metabolism, Starch and sucrose metabolism, Cell cycle, Spliceosome, and so on (Fig. 3D). Furthermore, we found three same pathways (Carbon metabolism, Starch and sucrose metabolism, and Ribosome) In DEPs and DCPs.

Metabolism regulation in developing fetal lung

As an important part of the living organism, metabolism is especially vital in fetal development. Cell adhesion, extracellular matrix tissue, vasculature development, and lipid metabolism are the main biological processes induced in early developmental stages. Defense/immune response, ion transport, and signal pathway had identified as the main biological processes induced in the late pregnancy [5]. We found that many metabolic pathways significantly down-regulate at the protein expression level (Fig. 4A). Such as carbon metabolism, glycolysis, TCA cycle, amino acid metabolism, and lipid metabolism, and so on. The level of crotonylation modification showed that it was significantly up-regulate in carbon metabolism and glycolysis (Figure S1A). Furthermore, we analyzed the relationship between proteins regulated in metabolism and MCODE through PPI (Fig. 4D, E, F) (Table 1). Hubba analysis can look the key protein. And by selected Degree and MCC Top10 through Hubba in Cytoscape (Fig. 4B, C), we found a total of 14 core proteins, of which 12 proteins have undergone regulation of crotonylation modification site (Table 2). These results indicate that with the development of the lung, the metabolism gradually increases, and the crotonylation modification may regulate the metabolism of the fetal lung.

Fig. 4

Metabolism regulation in Developing Fetal Lung. A KEGG functional enrichment analysis of down-regulated DEPs. B The Top 10 of Degree in PPI of metabolism of DEPs. (C) The Top 10 of MCC in PPI of metabolism of DEPs. D Metabolism of DEPs of Enrichment_GO_Color by cluster. E Metabolism of DEPs of Enrichment_GO_Color by PValue. F Metabolism of DEPs of PPI and MCODE by cluster

Table 1 Metabolism of DEPs of PPI and MCODE by clusterTable 2 Metabolism of DEPs of 12 proteins have undergone regulation of crotonylation modification siteGene expression regulation in developing fetal lung

Cell proliferation requires storage materials, and gene expression is an important part of cell proliferation and plays a decisive role in development. It had been reported that mRNA related to cell cycle, DNA repair/replication, RNA processing, and translation is the main biological process that decreases as the lung matures [5]. Our data show that Cell cycle, DNA replication, Homologous recombination, Mismatch repair, Base excision repair, Nucleotide excision repair, Basal transcription factors, RNA polymerase, Ribosome biogenesis in eukaryotes, and Ribosome are all significantly up-regulated (Fig. 5A). Meanwhile the level of crotonylation modification shows that the Cell cycle and Ribosome have enriched in DCPs (Figure S1B). Some protein sites was up-regulated, and some protein sites are down-regulated. The biological significance of crotonylation regulation needs to explore. Meanwhile we also found that the protein levels of 8 histones were up-regulated, and the crotonylation sites of 5 histones regulated to varying degrees. K55, K59, and K82 of H1.0 are down. K120 of H1.1 is up. K49, K78, and K100 of H1.5 is down, K55 is down. K47 of H1.10 is up, K75 reversly. K92 and K78 of H4 are down, K80 reversely (Fig. 5d). We further analyzed the proteins in the Cell cycle, and selected Degree and MCC Top5 through Hubba (Fig. 5B, C), and found a total of 8 core proteins, of which Cdk family proteins and Mcm proteins are the most. We found that there are more proteins of crotonylation in nuclear through subcellular localization, especially proteins related to chromatin, such as Crebbp and Mcm family proteins (Fig. 5E). However, crotonylation modification related to chromosome recombination and gene expression. Therefore, we speculate that protein crotonylation modification may affect lung development through cell proliferation.

Fig. 5

Gene Expression regulation in Developing Fetal Lung. A KEGG functional enrichment analysis of up-regulated DEPs. B The Top 5 of Degree in PPI of Cell cycle of DEPs. C The Top 5 of MCC in PPI of Cell cycle of DEPs. D Schematic diagram of gene expression of DEPs involved. E Schematic diagram of Cell cycle of DEPs and DCPs involved

Lung development regulation in developing fetal lung

As the fetus develops and grows, the lungs continue to develop and improve. We found 42 DEPs and 13 DCPs protein of GO Terms regulation in various parts of the lung through protein annotation (Tables 3 and 4). Lung epithelium development, lung cell differentiation, lung epithelial cell differentiation, lung alveolus development, lung secretory cell differentiation, lung morphogenesis, and epithelial tube branching involved in lung morphogenesis are more proteins regulation. There are also modifications in these biological processes. We found 24 and 14 terms of biological process related to lungs in DEPs and DCPs (Fig. 6A, B). Among them, the 41 DEPs and 13 DCPs are in the term of lung development. As a result, the four proteins (Hmgb1, Cux1, Itga3, and Asah1) have dysregulated in expression and crotonylated of protein. For example, in the nucleus, Hmgb1 is one of the major chromatin-associated non-histone proteins. It acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V(D)J recombination, DNA repair, and genome stability [10]. At the same time, we also found the regulation in transcription factor–for instance, Foxp1, Foxp4, Sox9, Nfib, Gata6, and Nkx2-1. Nfib, Gata6, and Nkx2-1 are participating in many lungs of GO terms. Regarding protein of crotonylation of the lung, Ctnnb1 and Yap1 of crotonylation protein are participating in many GO terms of lungs.

Table 3 Lung development regulation of 42 DEPs in developing fetal lungTable 4 Lung development regulation of 13 DCPs in developing fetal lungFig. 6

Lung Development regulation in Developing Fetal Lung. A, B GO enrichment analysis of lung related DEPs and DCPs, respectively. C KEGG functional enrichment analysis of (Notch, TGF-beta, and Wnt) signaling pathway DEPs and DCPs. D Schematic diagram of Notch signaling pathway and proteins up-regulated (red)

Six signaling pathways are involved in lung organogenesis, such as Notch, Tgfβ / Bmp, Sonic hedgehog (Shh), Fgf, Egf, and Wnt [11]. Studies have reported that the predicted inhibitory role of Notch signaling during lung maturation [5]. Our data is also significantly enriched in the Notch signaling pathway and up-regulate (Fig. 6D). In KEGG analysis, the 14 DEPs and 9 DCPs belong to the Wnt signaling pathway (Fig. 6C). Crebbp is only both dysregulated. The protein is up to expression and the position of K1524 occurred down in modification. Meanwhile, we found the 9 DEPs and 2 DCPs belong to Notch signaling pathway. Wherein, Crebbp is only both dysregulated. We also found the 11 DEPs and 7 DCPs belong to the Tgf-beta signaling pathway. The Crebbp and Dcn are both dysregulated. Therefore, Crebbp is the role of importance in these pathways; it maybe regulates lung development. In the PPI network of all the dysregulated proteins of expression, Crebbp interacts with many proteins. Maybe it can influence all the dysregulated proteins of expression. Hence, we suggest that Crebbp is a key protein of lung organogenesis; it can be a biomarker to detect lung development.