Proteomics confirms GLUT1 absence in CD34⁺ GLUT1-KO–derived reticulocytes and metabolite analyses reveals downregulated metabolic processes. (A) Simplified schematic of multiomics cell preparation, in which CD34⁺ from 3 donors were nucleofected with either GLUT1–targeting or NT sgRNAs, expanded and differentiated into reticulocytes; 10 million filtered reticulocytes were needed for comprehensive analyses of the proteome, metabolome, and lipidome. (B) Box plot comparing GLUT1 protein level between NT and GLUT1-KO reticulocytes as maximum label-free quantification (MaxLFQ) protein-level intensities. Box plot analysis (mean ± minimum to maximum with standard deviation) was performed by RStudio, and significance was calculated upon false discovery rate correction (∗∗∗P < .001). (C-D) Hierarchical clustering of the top 50 t test significant proteins (C) and metabolites (D) between NT and GLUT1-KO CD34⁺-derived reticulocytes. (E) Schematic representation of glycolysis, the polyol pathway, and the glutathione redox cycle in which proteins and metabolites are color-coded by log₂ (fold change) of GLUT1-KO reticulocytes in relation to NT control. The 10 steps of glycolysis are represented, with glucose and lactate both reduced in the KO whereas the remaining intermediate products increased. All involved enzymes are decreased (HK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK1, phosphofructokinase-1; TPI1, triosephosphate isomerase; BPGM, biphosphoglycerate mutase; PGK, phosphoglycerate kinase; PGAM, phosphoglycerate mutase; PK, pyruvate kinase; and LDHA, lactate dehydrogenase A). There is an imbalance in the glutathione cycle, as a consequence of increased reactive oxygen species (ROS), characterized by the depletion of reduced glutathione (GSH) and increase of oxidized glutathione (GSSG) and glutathione peroxidase 1 (GPX1). The hexose monophosphate (HMP) shunt is upregulated as a source of reduced NAD phosphate (NADPH), needed to maintain glutathione in its reduced form. An increase in polyols (such as sorbitol and mannitol) was also detected, which can be converted into fructose-1,6-phosphate by sorbitol dehydrogenase (SORD) and ketohexokinase (KHK). G6PD, glucose-6-phosphate dehydrogenase; PGLS, 6-phosphogluconolactonase; GSR, glutathione-disulfide reductase. Created with BioRender.