LC–MS/MS Method for Quantifying Glucose in Mammalian Brain Cells

Researchers from the Texas Tech University Health Sciences Center in Amarillo, Texas developed a new technique for quantifying glucose transporter 1 (GLUT1) using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Their findings, published in the Journal of Chromatography Open, focused on quantification in mammalian brain cells (1).

Staircase leading to the arena and its tower on the campus of Texas Tech University in the city of Lubbock | Image Credit: © jkgabbert - stock.adobe.com

Glucose is a major source of energy for mammalian cells, and due to its nature, glucose uptake by mammalian cells requires the use of dedicated transporters that facilitate its entry into cells. There are two major types of glucose transporters: those that are sodium-independent and facilitate glucose diffusion based on the chemical gradient (GLUTs), and those that are sodium-dependent and function as symport transporters (SGLTs). Among the tissues that use glucose as a major energy source, the central nervous system (CNS) likely represents the type of tissue that has the highest consumption rate adjusted by the tissue weight (20% of daily glucose uptake, for an average weight of 1.5–2 Kgs) (2).

Glucose uptake in the brain is rate-limited by the presence of a blood-brain barrier (BBB), a part of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) and pericytes ensheathed by a basement membrane and surrounded by astrocytic end-feet processes and neurons. As of today, using antibody-based biological assays to quantify GLUT1 expression remains the analytical technique used in literature. However, this approach remains challenging and limiting.

In this study, the scientists developed a new analytical method based around LC–MS/MS to provide specific and absolute GLUT1 quantification in human and non-human cells. A specific peptide signature, (TFDEIASGFR), was identified for GLUT1, with a retention time of 1.53 min with three distinct MRM transitions (571.8 > 894.3, 571.8 > 537.2 and 571.8 > 650.3 m/z ratios respectively). Following optimization, the method’s GLUT1 protein expression was compared to the immunoblot approach, which uses gel electrophoresis to detect proteins in a sample based on their size and electric charge (3). Overall, the scientists concluded their method was superior in terms of sensitivity (LLOQ=0.78 ng/mL vs. 3.125 μg/lane), better dynamic range (0.78–200 ng/mL versus 3.125–25 μg/lane) and better linearity (R2 = 0.999 versus R2 = 0.929) than the immunoblots counterpart. Additionally, their method was used to provide absolute GLUT1 quantification in various brain endothelial cells, showing differences in protein expression. Finally, they used the method to assess changes in GLUT1 protein levels during the differentiation of induced pluripotent stem cells (iPSCs) into astrocyte-like cells (iAstros) and brain microvascular endothelial cell-like cells (iBMECs).

The measured GLUT1 expression in the iBMECs was of 35.60 ± 0.80 fmol of GLUT1/µg of membrane protein. This value proved significantly higher than immortalized human and rodent BMECs, though it remained one-half to one-third of the GLUT1 protein levels reported in freshly isolated human brain micro vessels. This was expected, as the current limitation of iPSC differentiation protocol has yet to demonstrate the ability to obtain fully differentiated iBMECs regarding drug and solute transporters protein expression compared to their somatic cell counterparts. The method can help in assessing optimization of the differentiation protocol, by evaluating GLUT1 expression in differentiated iBMECs.

Finally, they investigated changes in GLUT1 expression through differentiation of their iPSC line into iAstros and iBMECs. Undifferentiated iPSCs showed a very high GLUT1 expression, though this was decreasing as cells went through differentiation. Undifferentiated iPSCs showed very high GLUT1 expression, though it decreased as cells underwent differentiation. Such decreases were inconsistent between differentiation protocols, as guided differentiation towards neuronal stem cell lineage caused a much-accentuated decrease.

Overall, the scientists’ comprehensive approach for absolute quantification of GLUT1 protein levels provided a notable alterative to tradition techniques. The limitations associated with antibody-based approaches were overcame, and the research contributed to a deeper understanding of GLUT1 expression in diverse cellular contexts. Future research could explore the functional consequences of GLUT1 dysregulation in different cell types, particularly regarding glucose uptake, metabolism, and cellular energetics.

References

(1) Mehta, Y.; Patel, D.; Perbaiz, I.; Bickel, U.; Al-Ahmad, A. J. Targeted Proteomics for Absolute Quantification of Glucose Transporter 1 in Mammalian Brain Cells Using Liquid Chromatography-Mass Spectrometry. J. Chromatogr. Open 2025, 7, 100198. DOI: 10.1016/j.jcoa.2024.100198

(2) Mergenthaler, P.; Lindauer, U.; Dienel, G. A.; Meisel, A. Sugar for the Brain: The Role of Glucose in Physiological and Pathological Brain Function. Trends Neurosci. 2013, 36 (10), 587–597. DOI: 10.1016/j.tins.2013.07.001

(3) Immunoblotting. ScienceDirect 2018. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/immunoblotting (accessed 2025-4-15)