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Proteomic analysis of human dentin matrix extracts by peptide-level high-pH reverse-phase fractionation

ABSTRACT: Proteomics research has increasingly focused on human cells, tissues, and fluids; however, comprehensive data regarding dental tissues remain limited. Dentin, a mineralized component of the tooth, contains structural proteins and bioactive molecules that, upon release, can modulate pulp cell activity and facilitate regeneration. Therefore, an understanding of the protein composition of dentin is of considerable importance. Previous studies have reported a limited number of proteins, and technical difficulties have often hindered replication. Furthermore, traditional extraction methods rely on strong acids, which are not clinically relevant. In this technical note, we introduce a workflow that combines EDTA-based dentin extraction under clinically relevant conditions with peptide-level fractionation using high-pH reverse-phase chromatography. This approach was compared against unfractionated samples (control), protein-level fractionation via SDS-PAGE, and peptide-level fractionation utilizing strong cation exchange (SCX), all followed by LC-MS/MS analysis. This workflow facilitated the identification of 514 proteins, compared with 238 (unfractionated control), 428 (SDS-PAGE), and 193 (SCX). High-pH reverse-phase chromatography contributed 217 unique identifications, surpassing the number of unique fractions identified through the other techniques. Although this methodology has been utilized in other protein systems, it has not previously been applied to dentin matrix extracts and represents a promising approach for enhancing the comprehensiveness of protein discovery.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Dentin

SUBMITTER: Bushra Gull

LAB HEAD: Mohamed Jamal

PROVIDER: PXD070849 | Pride | 2026-03-23

REPOSITORIES: Pride

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Project description:Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts, and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule is the enigmatic coupling factor. We have identified Cthrc1, a protein secreted by mature bone-resorbing osteoclasts, that targets stromal cells so as to stimulate osteogenesis. The expression of Cthrc1 is robustly induced when mature osteoclasts are placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increases in a high turnover state, such as that which is induced by RANKL injections in vivo, whereas it decreases with aging or following alendronate treatment, conditions associated with suppressed bone turnover. The targeted deletion of the Cthrc1 gene eliminates Cthrc1 expression in bone, whereas its deficiency in osteoblasts does not exert any significant effect. Osteoclast-specific deletion of the Cthrc1 gene results in osteopenia due to reduced bone formation: it also impairs the coupling process following resorption induced by RANKL injections, with a resultant impairment of bone mass recovery. Thus, Cthrc1 is an osteoclast-secreted “coupling factor” that regulates bone remodeling and hence, skeletal integrity. Total bone marrow cells were prepared from the femurs and tibias of 8-10-week-old C57BL/6 mice and cultured in the presence of M-CSF (100ng/ml) for 3 days as described previously (Takeshita et al., 2000 JBMR 15:1477-1488). Cells were harvested with 0.02% EDTA/PBS and used as bone marrow macrophages (BMMs). These BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (100ng/ml) for 2 days. TRAP positive mononuclear cells were harvested and used as pre-osteoclasts (pOC). These pOC cells were further cultured in the presence of M-CSF and RANKL for 2 days in normal plastic plate or on dentin slices. After 2 days, multinucleated TRAP positive mature osteoclasts were generated as mature osteoclasts on plate (mOCp) and mature resorbing osteoclasts on dentin (mOCd), respectively. RNAs were extracted from four different stages of osteoclast lineage cells; BMMs, pOC, mOCp and mOCd, and used for microarray analysis.

Project description:Dendritic cells (DCs) are the sentinels of the mammalian immune system and they undergo a complex maturation process mediated by activation upon pathogen detection. Recent studies described the analysis of activated DCs by transcriptional profiling, but translation regulation was never taken in account. Therefore, the nature of the mRNAs being translated at various stages of DC activation was determined with the help of translational profiling, which is the sucrose gradient fractionation of polysomal-bound mRNAs combined to microarrays analysis. Total and polysomal-bound mRNA populations were compared in immature (0h) and LPS-stimulated (4h and 16h) human monocyte-derived DCs with the help of Affymetrix microarrays. Biostatistical analysis indicated that 296 mRNA molecules are translationally regulated during DC-activation. The most abundant biological process among the regulated mRNAs was protein biosynthesis, indicating the existence of a negative feedback loop regulating translation. Interestingly, a cluster of 17 ribosomal proteins were part of the regulated mRNAs, indicating that translation may be fine-tuned by particular components of the translational machinery. Our observations highlight the importance of translation regulation during the immune response, and may favour the identification of novel gene clusters or protein networks relevant for immunity. Our study also provides information on the possible absence of correlation between gene expression and real protein production in DCs. To identify translationally regulated mRNA molecules, gene expression derived from the polysome-bound mRNAs was compared by Affymetrix microarrays analysis to the gene expression derived from unfractionated total mRNAs derived from whole-cell lysates, as recently described on several reports (Johannes 1999, Rajasekhar 2003, Bushell 2006, Lü 2006, Parent 2008). Polysomal RNA (P) and total RNA (T) were isolated from MoDCs generated from four different blood donors. Since three timepoints (0h, 4h and 16h) were chosen for each blood donor and RNA type, twenty-four RNA samples were totally analyzed by microarrays.

			Action	DRS
	DIA_Prot_50cm_EDTA_1.raw	Raw
	DIA_Prot_50cm_EDTA_2.raw	Raw
	DIA_Prot_50cm_EDTA_3.raw	Raw
	DIA_Prot_50cm_EDTA_4.raw	Raw
	DIA_Prot_50cm_EDTA_5.raw	Raw

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Proteomic analysis of human dentin matrix extracts by peptide-level high-pH reverse-phase fractionation

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