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Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS.

ABSTRACT: Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to (1) comprehensively characterize the human adipocyte proteome for the first time and (2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and high performance liquid chromatography-electron spray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from approximately 0.5 g of subcutaneous abdominal adipose tissue of three healthy, lean subjects, we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis, and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases.

SUBMITTER: Xie X

PROVIDER: S-EPMC2935302 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS.

Xie Xitao X Yi Zhengping Z Bowen Benjamin B Wolf Cassandra C Flynn Charles R CR Sinha Sandeep S Mandarino Lawrence J LJ Meyer Christian C

Journal of proteome research 20100901 9

Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to (1) comprehensively characterize the human adipocyte proteome for the first time and (2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and high performance liquid chromatography-electron spray ionization-tand ...[more]

PMID: 20812759

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Project description:We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase ? subunit (?-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, ?-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple ?-F1-ATPase peptides. There were three ?-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to ?-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R(2)?=?0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the ?-F1-ATPase(134-143) peptide. Measured stable-isotope enrichment was highly reproducible for the ?-F1-ATPase(134-143) peptide (CV?=?2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R(2)?=?0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the ?-F1-ATPase(134-143) peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle ?-F1-ATPase based on the determination of the enrichment of the ?-F1-ATPase(134-143) peptide.

Dataset Information

Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS.

Publications

Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS.

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