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

Area of Science:

• Proteomics
• Endocrinology
• Comparative Physiology

Background:

• Adipose tissue (AT) is a crucial endocrine organ implicated in evolution and disease.
• Proteomic analysis of AT is vital for understanding its diverse functions.
• Challenges in AT proteomics include high lipid and low protein content, and protein association with lipid droplets.

Purpose of the Study:

• To present a reproducible, cost-effective, gel-free, label-free proteomics protocol for adipose tissue.
• To overcome common challenges in adipose tissue proteomic analysis.
• To facilitate the study of adipose tissue in various species, including non-model mammals.

Main Methods:

• A gel-free, label-free, bottom-up, quantitative proteomics approach.
• Tissue homogenization, protein precipitation, and solubilization in SDC/TCEP buffer.
• Alkylation, in-solution tandem digestion (trypsin/Lys-C), SDC removal via acidification, followed by mass spectrometry.

Main Results:

• Reproducible protein and peptide identification rates achieved.
• Successfully identified over 1500 proteins in non-model mammal adipose tissue.
• Detected proteins relevant to comparative physiology, metabolism, and disease.

Conclusions:

• The developed protocol is robust, cost-effective, and minimizes processing time.
• It is accessible to researchers with varying technical expertise.
• This method advances the proteomic study of adipose tissue, aiding research in physiology and disease.