Lower Limit of Detection Advances Understanding of Metabolic Signaling in Human Muscle
Irene Tobias, Ph.D., is a Postdoctoral Researcher in the Biochemistry & Molecular Exercise Physiology Laboratory working with principal investigator Andy Galpin, Ph.D., at California State University, Fullerton’s Center for Sport Performance. Their research focuses on fiber type-specific metabolic signaling in human skeletal muscle, with the goal of understanding more about muscle physiology and the complex metabolic signaling pathways that contribute to health and human performance.
The Study of Fiber Type-specific Signaling Proteins
Human skeletal muscle tissue is a heterogeneous mixture of different fiber types (fast twitch, slow twitch and hybrids) that have vastly different metabolic properties. The composition of fiber types varies significantly between individuals and is highly dependent on exercise history, age and health status. However, most research on human muscle is performed with mixed fiber homogenates and neglects the dissection and consideration of different fiber types—mainly because the procedures involved are significantly more laborious and impractical. Individual fiber segments must be isolated and typed via myosin heavy chain expression. The isoforms of this highly abundant protein can be identified in single fibers using silver staining and relative molecular weight comparisons. However, the proteins that regulate metabolic signaling are far less abundant and typically below the limit of detection achieved with traditional Western blotting techniques. Irene and her group were faced with tissue-intensive and cumbersome procedures to accumulate enough pooled human muscle fibers from extracted biopsies for fiber type-specific protein measurements.
Enhanced Sensitivity with Wes Enables Single Muscle Fiber Segment Analysis
With Wes™, Irene can measure cell signaling proteins from a single muscle fiber segment. In fact, the group was able to lower the limit of detection nearly 20-fold compared with traditional Western blotting. Irene reports, “The enhanced sensitivity of Wes allows measurements of less abundant proteins to be feasible in single fiber samples, thus enabling robust study from far fewer typed fibers.” Irene concluded, saying, “Wes allows for much more precise, reproducible and faster results to be generated, in addition to far greater ease of operation, analysis and waste disposal. Overall, the laborious nature of previous methods is significantly reduced, which may ultimately encourage other investigators to employ Wes for fiber type-specific studies.”
Addressing Antibody Validity Concerns with Wes
Irene and her group recently published their findings in the Journal of Applied Physiology, in an article titled Fiber type-specific analysis of AMPK isoforms in human skeletal muscle: advancement in methods via capillary nano-immunoassay.
With an increased focus on antibody validity due to apparent molecular weight shifts observed with Wes, reviewers of the article requested additional experiments to prove antibody specificity during the manuscript review process. The observed molecular weight of a protein can differ between Western blotting and Wes as it depends on the matrix or gel type used, running buffers, characteristics of the target protein or even the ladder used—read our Technical Note on Molecular Weight Determination by Electrophoresis of SDS-denatured Proteins for a full discussion on the topic. With Wes, Irene and her team were able to quickly address their comments on apparent molecular weight changes by performing additional experiments to show that the antibodies were detecting the correct target of interest.
Advancing Muscle Metabolic Signaling Studies with Fiber Type-specific Protein Analysis
Key signaling events that control muscle physiology can be unresolvable in mixed fiber studies. The implementation of fiber type-specific protein analysis is advancing the field of muscle physiology by enabling the dissection of complex metabolic signaling pathways.