Protein Sample Preparation for Bottom-Up, Label-Free Quantitative Proteomics of Adipose Tissue

Adipose tissue (AT) is a complex, multifunctional endocrine organ that plays a significant role in animal evolution and human disease. Profiling of the proteome, or the set of proteins produced by a cell or tissue at a given time, can be used to explore the myriad functions of adipose tissue and understand its role in health and disease. The main challenges of adipose tissue proteomics include the high lipid and low protein content of the tissue and association of many proteins with lipid droplets. Here, we present a protocol for gel-free, label-free, bottom-up, relative quantitative proteomics of adipose tissue based on findings from the literature and our laboratory that yields reproducible protein and peptide identification rates while minimizing cost and processing time. This approach involves tissue homogenization, protein precipitation from homogenates, solubilization and denaturation of proteins in a buffer containing 5% sodium deoxycholate (SDC, an acid-insoluble detergent) and 5 mM tris(2-carboxyethyl)phosphine (TCEP, a reducing agent), alkylation with chloroacetamide, and in-solution tandem digestion with trypsin and Lys-C enzymes in the presence of 1% SDC. Acidification of peptides efficiently removes SDC prior to desalting and mass spectrometry. This method has been used successfully in our laboratory by both experienced researchers and those with limited technical backgrounds, including high school, undergraduate, and graduate students. We have identified >1500 proteins in adipose tissue of non-model mammals (e.g., blubber of marine mammals) spanning a dynamic range of 10⁵ using this approach, including proteins of interest for comparative physiology such as adipokines, metabolic and antioxidant enzymes, lipid droplet proteins, metabolite transporters, and mitochondrial proteins, among others.