Targeted proteomics for absolute quantification of glucose transporter 1 in mammalian brain cells using liquid chromatography-mass spectrometry

• We developed an LC-MS/MS method to quantify GLUT1 in rodent and human cells. • GLUT1 signature decapeptide (TFDEIASGFR) is specific, and not found in other GLUTs. • LC-MS/MS had better GLUT1 sensitivity than immunoblots. • iPSC-derived brain endothelial cells have higher expression than cell lines. • GLUT1 level decreases during differentiation into astrocytes and endothelial cells. Glucose represents a major source of energy for mammalian brains. Its uptake is tributary to a functional glucose transporter (GLUT1) expression at the blood-brain barrier (BBB). However, detecting and quantifying GLUT1 at the protein level using antibodies-based methods can be challenging. In this study, we have described an analytical method (using liquid-chromatography mass-spectrometry (LC-MS/MS)) to provide a specific and absolute quantification of GLUT1 in human and non-human cells. We identified a specific peptide signature for GLUT1 (TFDEIASGFR), 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, we compared the GLUT1 protein expression of our method versus immunoblot and concluded that our method was superior in terms of sensitivity (LLOQ=0.78ng/mL vs. 3.125μg/lane), better dynamic range (0.78–200ng/mL versus 3.125–25μg/lane) and better linearity (R2 = 0.999 versus R2 = 0.929) than the immunoblots counterpart. Furthermore, we used such a method to provide absolute GLUT1 quantification in various brain endothelial cells, showing differences in protein expression. Finally, we also used this 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). Taken together, we have developed and optimized a proteomic method for the absolute quantification of GLUT1 in mammalian cells, which allows an alternative method for protein quantification independent of the antibody-based ones.