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: