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J Biomol Tech. 2010 September; 21(3 Suppl): S10–S11.
PMCID: PMC2918207

Differential Protein Expression to the Hepatic Response from a High Fat Diet using Complementary Quantitative Approaches: 2D-DIGE, Label-Free and iTRAQ Labeling

K.C. Vickers,2 G. Wang,1 A.T. Remaley,2 and M. Gucek1
1Proteomics Core Facility, National Heart Lung and Blood Institute, Bethesda, MD, United States;
2Lipoprotein Metabolism Section, National Heart Lung and Blood Institute, Bethesda, MD, United States

Abstract

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Quantitative proteomic technologies have been developed to comprehensively identify and quantify proteins in two or more complex samples. Labeling techniques such as DIGE and iTRAQ are used to differentially label proteins or peptides, respectively. Label-free relative quantitation is an alternative approach that directly compares the peptide peak areas between LC–MS runs using an “in house” program QUOIL (quantification without isotope labeling). These complementary quantitative approaches were used to observe the hepatic responseto a typical Western diet rich in saturated fats. C57BL6 micewere fed a high fat diet (42% calories from fat) or normal chowfor 3 weeks. Dietary fat has been shown to cause substantial changes in hepaticlipid and lipoprotein metabolism. Rat liver homogenates were used for 2D gel separation and trypsinized for label-free and iTRAQ approaches. The iTRAQ labeled peptides were off-line separated by SCX chromatography followed by LC-MS/MS where 25 fractions were analyzed. The new dual-pressure trap, LTQ Orbitrap Velos mass spectrometer was used to profile the differentially expressed proteins, for both label-free and iTRAQ samples. Over 1300 proteins were identified using all three approaches with a false discovery rate of less than 5.0%. Of the proteins identified, over 400 proteins were differentially expressed 1.2 fold or more. Multiple amino acid metabolic pathways were significantly altered at the protein level (cysteine/methionine and taurine metabolism). We were able to integrate some of the proteomics data with previously obtained genomic and metabolomic datasets. In the high fat sample, the enzyme cysteine sulfinic acid carboxylase decreased in the mRNA and protein data, 5.97 and 2.06 fold respectively. The resulting metabolite taurine was down regulated 37 fold in previous metabolic studies. In summary, the use of a complementary approach enabled the identification of many differentially expressed proteins involved in the intricate hepatic response to dietary fat.


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