Quantifying GLUT1 in Mammalian Brain Cells
Hello and welcome to Science with Sandra!
I hope everyone is having a great start to this new year!
As we begin this new year, I would like to highlight a recent publication by Dr. Abraham Al-Ahmad and his team at Texas Tech, “Targeted proteomics for absolute quantification of glucose transporter 1 in mammalian brain cells using liquid chromatography-mass spectrometry”. This publication emphasizes the significance of having reliable techniques in the laboratory setting to quantify protein expression. The importance of having reliable techniques in the laboratory is key to having trustworthy experimental results, which can then translate into dependable diagnostic techniques and reliable tests that can evaluate treatments’ effectiveness.
Dr. Al-Ahmad and his team start their publication by describing that detection and quantification of the GLUT1 protein using antibody based methods can be very challenging. Antibodies are protective proteins produced by the immune system that attach to foreign substances to help remove them from the body. In research laboratories, different techniques utilize antibodies to detect other proteins, however many commercially available antibodies and antibody conjugates are not well characterized and lack validation. Some of the difficulties of using antibodies for the detection of GLUT1, according to the authors, are the apparent variations in molecular weights of GLUT1 across cell types. The authors emphasize that all of these issues highlight the importance of developing analytical methods that allow specific detection and quantification of GLUT1 independent of antibodies.
The publication by Dr. Al-Ahmad and his team describes the development of an analytical method using liquid-chromatography mass-spectrometry (LC-MS/MS) which provides a specific and absolute quantification of GLUT1 in human and non-human mammalian cells. Importantly, they identified a specific decapeptide signature, TFDEIASGFR (each of the letters represent an amino acid) for the GLUT1 protein that is part of the C-terminal tail of the protein which is close to the 12th transmembrane domain. (See figure below).
Pascual, J et al 2004
COOH represents the C-terminal tail of the protein. The signature peptide identified by Dr. A-Ahmad and his team is located in this tail.
The study team validated their method against other methods used to determine protein expression such as immunoblots. Their results showed that their method provides an absolute quantitative method for GLUT1 protein expression and appears to be more precise. Interestingly, their method identified different expression patterns of the GLUT1 protein and showed that undifferentiated induced pluripotent stem cells (iPSCs) had the highest expression levels of GLUT1 protein compared to other cells tested including brain endothelial cells, which are considered one of the cell types with the highest expression of GLUT1.
Finally, Dr. Al-Ahmad and his team provide some insights regarding the study limitations. They mentioned that this study focused mainly on brain endothelial cells lines and that although these cells provide invaluable insights into GLUT1 expression in the blood brain barrier, they may not reflect the full complexity of the GLUT1 expression in the entire brain. In addition, they emphasize the importance of performing more studies that will incorporate primary cell cultures and/or in vivo models to provide a more complete picture of the GLUT1 expression in different models.
We thank Dr. Al-Ahmad and his team for developing models and techniques that facilitate the study of the GLUT1 protein and that allow to have a better understanding of how this protein’s disruption affect GLUT1 Deficiency patients. Thank you for visiting our blog and please, do not hesitate to contact me at [email protected] if you have any questions.
