Integrative Proteomics and Phosphoproteomics Profiling of Symptomatic Accessory Navicular Bone Based on Tandem Mass Tag Technology

Abstract

Background: The accessory navicular bone (ANB) is a common accessory bone in the foot. Certain ANBs significantly impair patients' feet normal walking function. Foot injury is associated with ANB after athletic training. However, the molecular mechanism of foot injury with ANB after athletic training remains unclear. This study aims to investigate the proteomics and phosphoproteomics profile of foot injury with the ANB after athletic training.

Patients and methods: We collected ANB tissues and normal bone (NB) tissues from 5 foot injury patients with ANB after 3 months of athletic training to perform proteome sequencing by tandem mass tag (TMT) technology. Then, the differentially expressed proteins (DEPs) and phosphorylation proteins (DPPs) were identified between the ANB and NB groups. Furthermore, the potential functions of DEPs and DPPs were annotated, respectively. Besides, the protein-protein interaction (PPI) network was constructed for DEPs.

Results: A total of 147 DEPs (129 upregulated and 18 downregulated) were detected. Functional enrichment suggested that they were involved in extracellular matrix (ECM)-receptor interaction and cell adhesion. PPI network showed that COL4A1 and COL4A2 had the highest interaction score, followed by RBBP4 and RBBP7. In addition, phosphoproteomics analysis identified 4 upregulated and 1 downregulated DPPs, and they were primarily enriched in regulating lipolysis in adipocytes.

Conclusion: Our study found that foot injury with ANB after exercise training may be associated with proteins related to inflammation and immunity (such as MRC1, UBE2N, CYCS), bone repair and regeneration (such as Emilin2, COL4A1, COL4A2, and ITGA9), and bone microstructure homeostasis (such as GCA and ANXA3). This provides new insights into understanding its pathogenesis and guiding treatment strategies.

Keywords: accessory navicular bone; athletic training; phosphoproteomics; proteomics.

Figure 1

ANB foot anatomy map and subtype classification. (A) Schematic diagram of ANB foot anatomy. (B) Schematic diagram of ANB subtypes.

Figure 2

DEPs between the ANB and NB group. (A) DEPs were displayed by volcano plots. Red dots represent up-regulated DEPs, and green dots represent down-regulated DEPs. (B) DEPs were displayed by Heat map. Changes of the color from blue to red indicate that the DEPs are up-regulated. NB, normal bone; ANB, accessory navicular bone.

Figure 3

GO and KEGG pathways analysis for DEPs. (A) The bar chart showing the top ten biological processes, cellular components, and molecular functions. The vertical axis represents the GO terms, while the horizontal axis indicates the enrichment of DEPs in the corresponding functional terms. The larger of the -log10 P-value, the more correlated between the DEPs and function. (B) A bubble chart showing the top 15 KEGG pathways for DEPs. The bubble size represents the number of DEPs, and the bubble color represents the p value.

Figure 4

PPI network of DEPs. Nodes in the network represent proteins, lines represent interactions between them, and there is no known interaction between unconnected protein nodes. The wider the line, the higher the score.

Figure 5

Identified phosphorylation site. (A) Bar chart shows the number of phosphorylation sites mainly occurring on three types of amino acids. (B) Bar chart shows the relationship between different phosphosites number and the number of phosphopeptides. S, serine; T, threonine; Y, tyrosine.

Figure 6

Identification of DPPs between the ANB and NB group and functional enrichment. (A) DPPs were displayed by volcano plots. Red dots represent up-regulated DPPs, and green dots represent down-regulated DPPs. (B) A bubble chart showing the top 5 KEGG pathways for DPPs. The bubble size represents the number of DPPs, and the bubble color represents the p value.

Figure 7

KEGG pathway diagram showing regulation of lipolysis in adipocytes. The red frames represent DPPs and the green frames represent specific genes or enzymes.