Evaluation of 2-D DIGE for skeletal muscle: protocol and repeatability

Area of Science:

• Proteomics
• Biochemistry
• Human Skeletal Muscle Physiology

Background:

• Proteomic analysis generates extensive data but is often limited by quantification errors from gel-to-gel variability.
• Internal standards are crucial for reducing variations in proteomic quantification.

Purpose of the Study:

• To establish a robust sample preparation protocol for human skeletal muscle using two-dimensional difference gel electrophoresis (DIGE).
• To assess the repeatability and reliability of the Ettan DIGE system for proteomic analysis.

Main Methods:

• Developed a DIGE sample preparation protocol using minimal human skeletal muscle tissue (0.5 mg).
• Investigated Ettan DIGE system repeatability by labeling nine homogenate aliquots with different CyDye dyes (Cy2, Cy3, Cy5).
• Analyzed 18 x 24 cm gels using Typhoon 9410 laser scanner and DeCyder software.

Main Results:

• Achieved separation of over 2500 distinct protein spots across a pH range of 3-11 and molecular weight of 10-200 kDa.
• Demonstrated high repeatability with a mean error of 1.7% for triplicate spots (n=1314).
• Detected changes in protein abundance below 20% with 99% confidence, showing no false positives.

Conclusions:

• The developed DIGE protocol is efficient, requiring minimal tissue and offering high-resolution protein separation.
• The Ettan DIGE system provides a repeatable and accurate method for quantitative proteomic analysis of human skeletal muscle.
• This method is valuable for comparing protein abundance across different physiological or pathological conditions.