Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

Orphanet Journal of Rare Diseases

Takeshi Tomonaga²  Shio Watanabe²  Jun Adachi²  Yuuki Hashimoto²  Ken-ichi Hirano¹  Naoko Kawasaki²  Yasuhiro Hara² 

[1] Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan;Laboratory of Proteome Research, National Institute of Biomedical Innovation 7-6-8 Saito-Asagi, Ibaraki City, Osaka 567-0085, Japan

关键词: Rare disease;    ATGL;    SRM/MRM;    SILAC;    Triglyceride deposit cardiomyovasculopathy;    Proteome;   

Others  :  863339
DOI  :  10.1186/1750-1172-8-197

 received in 2013-09-24, accepted in 2013-12-12,  发布年份 2013

【 摘 要 】

Background

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis.

Methods

To identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells.

Results

Using SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search.

Conclusions

We performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future.

【 授权许可】

   
2013 Hara et al.; licensee BioMed Central Ltd.

【 图 表 】

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