Abstract
The tooth is one of the ideal models for developmental study, involving in epithelial-mesenchymal transition and cell differentiation. The essential factors and pathways identified in tooth development will help understand the natural development process and the malformations of mineralized tissues such as skeleton. The time-dependent proteomic changes were investigated by healthy human molars proteomics of embryonic stages from the cap-to-early bell stage. A total of 713 differentially expressed proteins (DEPs) with five temporal expression patterns were filtered. 24 potential driver proteins of tooth development were screened by weighted gene co-expression network analysis (WGCNA) including CHID1, RAP1GDS1, HAPLN3, AKAP12, WLS, GSS, DDAH1, CLSTN1, AFM, RBP1, AGO1, SET, HMGB2, HMGB1, ANP32A, SPON1, FREM1, C8B, PRPS2, FCHO2, PPP1R12A, GPALPP1, U2AF2 and RCC2. The hub proteins in different temporal expression patterns were extracted. And the potential cell resources and the temporal expression patterns at transcriptomic level were explored using single cell RNA-sequencing (scRNA-seq). This study provides invaluable resources for the mechanistic studies of human embryonic epithelial and mesenchymal cell differentiation and tooth development.
Keywords: Human deciduous molar; label-free proteome; temporal expression pattern; stem cell differentiation
Introduction
The tooth is one of the ideal models for developmental studies, including the interactions between dental epithelium and mesenchyme, cell differentiation, tissue mineralization, maturation, and tooth eruption[1]. The tooth is also an ideal source for stem cells. Although adult human tooth have been widely used for isolating dental stem cells, the self-renewal dental epithelial stem cells or highly proliferative dental mesenchymal cells only exist in the embryonic phase of tooth development[2]. Therefore, the study of tooth development is helpful to understand stem cell-driven organ regeneration and promote the treatments for dental patients. Although much has been learned from in vitro cultured embryonic stem cells, the biological characteristics were inevitably changed along with cell culture. Thus, the study of natural physiological process of tooth germ will provide new information for understanding the tooth development.
The applications of multi-omics methods have provided researchers with the comprehensive understanding of natural tooth development[3, 4]. Shi Y and colleagues applied single cell RNA-sequencing (scRNA-seq) to identify the molecular pathways in immature human tooth germ[5]. Recently, Alghadeer A and colleagues contributed a scRNA-seq dataset for identifying the development of human embryonic tooth germ[6]. However, the post-transcriptional biological processes such as gene modification and protein translation also play important roles in organ development.
Proteins are the important participants in the execution of intra- and intercellular biological activities of linking genotypes and phenotypes[7]. But there have been only few proteomics-related studies on human embryonic tooth development. Herein we performed proteomics profiling on the whole tooth germ from 15 healthy donors of the post-conception weeks (PCW) 11-13, 14-16, and 17-19 to identify molecular networks associated with tooth morphogenesis. In addition, the public scRNA-seq datasets were applied to compare the temporal pattern between protein and RNA expression. The temporal expression patterns of some proteins were selectively validated by immunohistochemistry analyses. This study provides an invaluable resource for temporal protein expressions of human tooth in natural conditions.
Materials and methods
Tissue collection and ethics approval
Fifteen healthy human embryonic tooth germ tissues were used in the study, with each providing two mandibular molars. The post-conceptional ages were estimated using ultrasonography and grouped as: PCW 12±1, 15±1, and 18±1. All the samples were collected after voluntary artificial abortion and excluded from detectable abnormalities recognized by ultrasound or genetic sequencing. Written informed consents were obtained from the all the pregnant women. Written informed consents were obtained from the all the pregnant women. Permission for this study was authorized by the Ethics Committee of Longgang District Maternity&Child Healthcare Hospital of Shenzhen City (LGFYYXLLQ-2021-005).
Tissue protein extraction and digestion
Each sample was ground into powder with liquid nitrogen and then added 5 times volume of lysis buffer containing 1% Triton X-100 and 1% protease inhibitor cocktail. Next, the high-intensity ultrasonic processor (Scientz, China) was used for ultrasonication 3 times. The supernatant was acquired after centrifugation. BCA kit (Beyotime, China) was used for protein quantification. Then the supernatant was precipitated by acetone (5 times volume) at the temperature of -20℃. The precipitate was ultrasonic suspended with 200 mM triethylammonium bicarbonate. 2% volume of the trypsin was used for digestion. Finally, 5 mM dithiothreitol was added for 30 min at the temperature of 56 °C and 11 mM iodoacetamide was used for alkylated at room temperature avoiding exposure of light for 15 min .
4-Dimensional label-free mass spectrometer liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis
The tryptic peptides were dissolved in 0.1% formic acid and 2% acetonitrile aqueous solution and uploaded onto a lab-made analytical column (Hangzhou Jingjie PTM BioLab). Peptides were separated with a serial of acetonitrile containing 0.1% formic acid for 64 min, 23% to 32% for 18 min and 80% for 4 min using EASY-nLC1200 nano ultra-high performance LC system (Thermo Fisher) with a 500 nL/min flow rate.
The peptide segments were analyzed by the Orbitrap ExplorisTM 480 (Thermo Fisher) mass spectrometry. The electrospray voltage was set as 2.3 kV and the MS scan range of the Orbitrap detector was set from 400 to 1200 m/z. The data-dependent scanning (DDA) mode data were acquired. The dynamic exclusion was set to 20 s.
Database search and protein quantification
The MaxQuant search engine (version 1.6.15.0) was used for the raw MS data processing. The tandem mass spectra were searched against UniProtKB/Swiss-Prot database. Less than 2 missing cleavages in trypsin/P were allowed for cleavage enzyme identification. The mass tolerance for precursor ions was set to 10 ppm and the fragment ions was 0.02 Da. In this study, the fixed modification was carbamidomethyl on Cys, the variable modifications include oxidation on Met, acetylation on protein N-terminal, and deamidation on protein N and Q. The false discovery rate (FDR) was adjusted to < 0.01. The identified protein must include one or more unique peptides. The relative quantitative value (R) of each protein of all the samples was calculated by the label-free quantification intensity (I) centrally transformation, according to: