TY - JOUR
T1 - A method for the gross analysis of global protein acylation by gas–liquid chromatography
AU - Mohammadzadeh, Fatemeh
AU - Hosseini, Vahid
AU - Mehdizadeh, Amir
AU - Dani, Christian
AU - Darabi, Masoud
N1 - Funding Information:
The research was financially supported by a grant (grant number, 273) from the Liver and Gastrointestinal Diseases Research Center of Tabriz University of Medical Sciences. The authors would like to thank the National Council for Development of Stem Cell Sciences and Technologies for supporting this work. We are also thankful to Maghsod Shaaker for his support and help during this work.
Publisher Copyright:
© 2018 International Union of Biochemistry and Molecular Biology
PY - 2019/3
Y1 - 2019/3
N2 - Protein acylation is a posttranslational modification in which an amino acid residue of a protein is acylated by a fatty acid. This process plays a key role in regulating proteomic function. Studies of protein acylation have relied on the development and application of extremely complicated molecular methods. However, global protein acylation can be profiled following hydrolysis of fatty acyl groups from cellular proteins. The present study aimed to develop a method for analysis of global protein acylation using gas–liquid chromatography (GLC). The total protein was extracted from the human hepatocellular carcinoma (HepG2) cell line. Protein sedimentation and extensive wash were combined with differential O-, S-, or N-acyl hydrolysis using sodium hydroxide (NaOH), hydroxylamine (NH 2 OH), or hydrochloric acid (HCl), respectively. GLC with a flame ionization detector system was used to analyze changes in the fatty acid composition of the released lipids. The effect of selective inhibition of monounsaturated fatty acid (MUFA) synthesis on global protein acylation and the expression of reprogramming markers were determined to further validate the proposed profiling approach. In all hydrolysis conditions, the amount of myristate released was significantly higher than of other fatty acids. Notable differences were observed in the release of individual fatty acids among the hydrolyzing agents. Only NH 2 OH could release significant amounts of palmitoleate (>2.5-fold vs. NaOH and HCl). The acylation assay indicates that treatment with a chemical inhibitor of monounsaturated fatty acid synthesis led to an overall increase in saturated fatty acid O- and N-acylation, and a decrease in palmitoleate O- and S-acylation of cellular proteins (<−15%). This was accompanied by significant reductions in the gene expression of the reprogramming markers Oct4 (−26%, P < 0.01) and Sox2 (−40%, P < 0.01). GLC-based analysis of global protein acylation affords a semi-quantitative method that can be used to assess the gross changes in the protein acylation profile during cell differentiation and reprogramming.
AB - Protein acylation is a posttranslational modification in which an amino acid residue of a protein is acylated by a fatty acid. This process plays a key role in regulating proteomic function. Studies of protein acylation have relied on the development and application of extremely complicated molecular methods. However, global protein acylation can be profiled following hydrolysis of fatty acyl groups from cellular proteins. The present study aimed to develop a method for analysis of global protein acylation using gas–liquid chromatography (GLC). The total protein was extracted from the human hepatocellular carcinoma (HepG2) cell line. Protein sedimentation and extensive wash were combined with differential O-, S-, or N-acyl hydrolysis using sodium hydroxide (NaOH), hydroxylamine (NH 2 OH), or hydrochloric acid (HCl), respectively. GLC with a flame ionization detector system was used to analyze changes in the fatty acid composition of the released lipids. The effect of selective inhibition of monounsaturated fatty acid (MUFA) synthesis on global protein acylation and the expression of reprogramming markers were determined to further validate the proposed profiling approach. In all hydrolysis conditions, the amount of myristate released was significantly higher than of other fatty acids. Notable differences were observed in the release of individual fatty acids among the hydrolyzing agents. Only NH 2 OH could release significant amounts of palmitoleate (>2.5-fold vs. NaOH and HCl). The acylation assay indicates that treatment with a chemical inhibitor of monounsaturated fatty acid synthesis led to an overall increase in saturated fatty acid O- and N-acylation, and a decrease in palmitoleate O- and S-acylation of cellular proteins (<−15%). This was accompanied by significant reductions in the gene expression of the reprogramming markers Oct4 (−26%, P < 0.01) and Sox2 (−40%, P < 0.01). GLC-based analysis of global protein acylation affords a semi-quantitative method that can be used to assess the gross changes in the protein acylation profile during cell differentiation and reprogramming.
UR - http://www.scopus.com/inward/record.url?scp=85057853390&partnerID=8YFLogxK
U2 - 10.1002/iub.1975
DO - 10.1002/iub.1975
M3 - Journal articles
C2 - 30501005
AN - SCOPUS:85057853390
SN - 1521-6543
VL - 71
SP - 340
EP - 346
JO - IUBMB Life
JF - IUBMB Life
IS - 3
ER -
