Temporal profiling of the secretome during adipogenesis in humans

Abstract

Adipose tissue plays a key role as a fat-storage depot and as an endocrine organ. Although mouse adipogenesis has been studied extensively, limited studies have been conducted to characterize this process in humans. We carried out a temporal proteomic analysis to interrogate the dynamic changes in the secretome of primary human preadipocytes as they differentiate into mature adipocytes. Using iTRAQ-based quantitative proteomics, we identified and quantified 420 proteins from the secretome of differentiated human adipocytes. Our results revealed that the majority of proteins showed differential expression during the course of differentiation. In addition to adipokines known to be differentially secreted in the course of adipocyte differentiation, we identified a number of proteins whose dynamic expression in this process has not been previously documented. They include collagen triple helix repeat containing 1, cytokine receptor-like factor 1, glypican-1, hepatoma-derived growth factor, SPARC related modular calcium binding protein 1, SPOCK 1, and sushi repeat-containing protein. A bioinformatics analysis using Human Protein Reference Database and Human Proteinpedia revealed that of the 420 proteins identified, 164 proteins possess signal peptides and 148 proteins are localized to the extracellular compartment. Additionally, we employed antibody arrays to quantify changes in the levels of 182 adipokines during human adipogenesis. This is the first large-scale quantitative proteomic study that combines two platforms, mass spectrometry and antibody arrays, to analyze the changes in the secretome during the course of adipogenesis in humans.

Figure 1

Differentiation of human preadipocytes to mature adipocytes. (A) Representative bright field images of subcutaneous fat depot-derived preadipocytes (Day 0) and day-6, 9, 12 differentiated adipocytes. (B) Proteins in the conditioned media were resolved by SDS-PAGE and visualized by silver staining.

Figure 2

iTRAQ-based quantitative proteomics. In the course of differentiation from human preadipocytes to adipocytes, conditioned media were harvested, desalted, and concentrated. The protein samples were subjected to reduction, alkylation, and tryptic digestion. After labeling with iTRAQ labeling reagent, the resulting peptides were mixed and fractionated by strong cation exchange liquid chromatography. Each fraction was analyzed by LC−MS/MS on an LTQ-Orbitrap Fourier transform mass spectrometer.

Figure 3

MS/MS spectra of iTRAQ labeled peptides from representative differentially secreted proteins identified in this study. (A) Adiponectin; (B) Lactotransferrin; (C) dimethylarginine dimethylaminohydrolase 1; and (D) Cathepsin B. (Insets) Reporter ions generated during MS/MS that were used for quantitation.

Figure 4

Dynamic expression patterns of proteins identified from the secretome of adipocytes during the course of differentiation. (A−C) Examples of proteins belonging to these distinct patterns.

Figure 5

Expression patterns of representative proteins commonly quantitated by mass spectrometry and antibody arrays. (A) Adiponectin; (B) Adipsin (Complementary Factor D); (C) Apolipoprotein E; (D) Cystatin C; (E) Matrix metalloproteinase 2; and (F) Osteonectin. In each panel, iTRAQ-based quantitation is shown on the left while antibody array-based quantitation is shown on the right.