Multiplexing of TMT labeling reveals folate-deficient diet-specific proteome changes in NTDs

Pei Pei; Jinying Shen; Xuejia He; Yubing Zeng; Ting Zhang; Shan Wang

doi:10.3389/fcell.2024.1294726

Multiplexing of TMT labeling reveals folate-deficient diet-specific proteome changes in NTDs

Front Cell Dev Biol. 2024 Mar 13:12:1294726. doi: 10.3389/fcell.2024.1294726. eCollection 2024.

Authors

Pei Pei¹, Jinying Shen¹, Xuejia He², Yubing Zeng³, Ting Zhang^{1

2

3}, Shan Wang^{1

2

3}

Affiliations

¹ Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, China.
² Capital Institute of Pediatrics-Peking University Teaching Hospital, Beijing, China.
³ Children's Hospital Capital Institute of Pdiatrics, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Abstract

Introduction: In the early stage of embryonic development, the neural tube (NT) cannot be closed properly due to some complex factors, including environmental factors, genetic factors, and the relationship between various factors, leading to the occurrence of neural tube defects (NTDs). Methods: In this study, we induced a mouse model of NTDs by feeding mice with a low-folate diet and intraperitoneally injecting them with 1.5 mg/kg methotrexate on E7.5. Fetal mice were achieved at E13.5, and we extracted proteins from brain tissues with trypsin digestion. After enzymatic digestion, peptides were labeled with TMT/iTRAQ and separated in high-performance liquid chromatography (HPLC) for subsequent liquid chromatography tandem mass spectroscopy (LC-MS/MS) analysis. We used gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation to analyze proteomic changes and analyze the functional enrichment of differentially expressed proteins (DEPs) in the NTD mice tissues. Results: A low-folate-induced mouse model was successfully constructed. Folate was used as a sensitizing agent, and the teratogenicity rate of the NTD fetal mice increased to 36.5% when the concentration of methotrexate was at 1.5 mg/kg. Mass spectrometry was used to identify 6,614 proteins, and among them, 5,656 proteins were quantified. In the following proteomic analysis, GO classification and KEGG pathway enrichment analysis were conducted, and heatmaps were drawn for differentially expressed proteins (DEPs). The main pathways associated with NTDs, such as the Hedgehog, Wnt, p53, and Hippo signaling pathways and the one-carbon pool mediated by folate, can be identified through a protein-protein interaction (PPI) network. It was also found that the regulation of ribosomal proteins, such as RPL13 and RPL14, which are upregulated in NTDs, has a certain impact on neural tube development. Discussion: Our results revealed proteomic changes in the tissues of low-folate-induced NTD mice. Validation showed that ribosomal proteins play a regulatory role during the development of NTDs and provides new ideas for the pathogenesis and preventive measures of NTDs.

Keywords: RPL13; RPL14; folate deficiency; neural tube defects; neural tube development; proteomics; ribosome.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the National Natural Science Foundation of China (No. 82071690). Beijing Municipal Natural Science Foundation (7242014), the Beijing Hospitals Authority’s Ascent Plan (DFL20221102), the Public Service Development and Reform Pilot Project of Beijing Medical Research Institute (BMR2021-3).