Extended Multiplexing of Tandem Mass Tags (TMT) Labeling Reveals Age and High Fat Diet Specific Proteome Changes in Mouse Epididymal Adipose Tissue

Deanna L Plubell,Phillip A Wilmarth,Yuqi Zhao,Alexandra M Fenton,Jessica Minnier,Ashok P Reddy,John Klimek,Xia Yang,Larry L David,Nathalie Pamir

doi:10.1074/mcp.m116.065524

Deanna L Plubell, Phillip A Wilmarth + Show 8 more

Open Access

https://doi.org/10.1074/mcp.m116.065524

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Abstract

The lack of high-throughput methods to analyze the adipose tissue protein composition limits our understanding of the protein networks responsible for age and diet related metabolic response. We have developed an approach using multiple-dimension liquid chromatography tandem mass spectrometry and extended multiplexing (24 biological samples) with tandem mass tags (TMT) labeling to analyze proteomes of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short or a long-term, and from mice that aged on low versus high fat diets. The peripheral metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet and advancing age, with long-term high fat diet exposure being the worst. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g. AACS, ACOX1, ACLY) and red-ox pathways (e.g. CPD2, CYP2E, SOD3) were significantly altered (FDR < 10%). Long-term high fat diet significantly altered 55 proteins associated with immune response (e.g. IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g. OTC, ARG1, CPS1), and amino acid biosynthesis (e.g. GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g. ARG1, CPS1), immune response/complement activation (e.g. C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g. EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g. YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Using our adipose tissue tailored approach we have identified both age-related and high fat diet specific proteomic signatures highlighting a pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. Data are available via ProteomeXchange with identifier PXD005953.

Highlights

From the ‡Department of Medicine, Knight Cardiovascular Institute, Oregon Health & Sciences University, Portland, Oregon; §Proteomics Shared Resources, Oregon Health & Sciences University, Portland, Oregon; ¶Department of Integrative Biology and Physiology, University of California, Los Angeles, California
We adjusted each sample to the average total reporter ion intensity within each tandem mass tags (TMT) experiment to control for small differences in peptide concentrations and labeling efficiencies, both by adjusting volumes of each TMT labeling reaction prior to the 2D LC-MS/MS analysis and afterward in software during analysis steps
We developed a novel normalization method to correct for the random sampling of parent ions across multiple LCMS/MS runs that placed each protein on a common reporter ion intensity scale across all three TMT experiments

Summary

Introduction

From the ‡Department of Medicine, Knight Cardiovascular Institute, Oregon Health & Sciences University, Portland, Oregon; §Proteomics Shared Resources, Oregon Health & Sciences University, Portland, Oregon; ¶Department of Integrative Biology and Physiology, University of California, Los Angeles, California. Using our adipose tissue tailored approach we have identified both age-related and high fat diet specific proteomic signatures highlighting a pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. The obesityrelated biological and metabolic changes within abdominal white adipose tissue could elucidate its role in the etiology of obesity related metabolic complications. The rapid spread of obesity and overweight has prompted efforts to understand the biological and metabolic dynamics of white adipose tissue in response to western living. White adipose tissue protein changes in response to duration of high fat diet and to maturation on either low or high fat diet are still not well understood.

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