Rat liver ESI-MS/MS
one-carbon metabolism in the liver plays a critical role in placental and fetal growth. Impaired functioning of one-carbon metabolism is associated with increased homocysteine (Hcy) levels. In this study, we applied a comprehensive proteomic approach to identify differential expression of proteins related to one-carbon metabolism in the livers of rat offspring as an effect of maternal food restriction during gestation. We determined that betaine-homocysteine S-methyltransferase 1 (BHMT1), methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), and ATP synthase subunit beta mitochondrial (ATP5B) expression levels were significantly reduced in the livers of rat offspring exposed to maternal food restriction during gestation compared with in the offspring of rats fed a normal diet (p<0.05).
Sample Processing Protocol
Approximately 0.2 g of frozen liver were prepared in lysis buffer on ice. . Each sample was sonicated for 30 s and centrifuged at 14 000 rpm for 30 min at 4°C. The supernatant was collected and the protein concentration was determined using a BCA protein assay kit (Sigma, St. Louis, MO, USA). Equal amounts of lysate, 250 µg was used for the 2-DE.
Data Processing Protocol
MS/MS was assigned as the ion search option in MASCOT software (v2.4, Matrix Science, London, UK). Peptide fragment files were obtained from the peptide peaks in ESI-MS by ESI-MS/MS. Trypsin was selected as the enzyme with one potentially missing cleavage site. ESI-QTOF was selected as the instrument type. The peptide fragment files were searched based on the database by using the MASCOT search engine [Database: NCBInr20100708 20100716 (11411477 sequences; 3893939158 residues) Timestamp: 31 May 2011] and limited to Rattus norvegicus.
You YA, Lee JH, Kwon EJ, Yoo JY, Kwon WS, Pang MG, Kim YJ. Proteomic analysis of one-carbon metabolism-related marker in liver of rat offspring. Mol Cell Proteomics. 2015 Sep 4. pii: mcp.M114.046888 PubMed: 26342040