Updated project metadata. Astrocytes and neurons form a highly specialized functional unit and the loss or gain of astrocytic functions can influence the initiation and progression of different neurodegenerative diseases. Neurons depend on the antioxidant protection provided by neighboring astrocytes. Glutathione (-l-glutamyl-l-cysteinyl-glycine) is a major component of the antioxidant system that defends cells against the toxic effects of reactive oxygen/nitrogen species. A decline in glutathione levels has been observed in aging and neurodegenerative diseases and it aggravates the pathology in an amyotrophic lateral sclerosis-mouse model. Using a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic approach, we analyzed the changes in global protein expression and regulation of lysine acetylation, in primary astrocyte cultures obtained from wild type mice or those deficient in the glutamate-cysteine ligase modifier subunit (GCLM). GCLM knockout astrocytes display an approximately 80% reduction in total glutathione levels. We identified potential molecular targets that are affected by the chronic decrease in glutathione levels and observed an adaptative response mediated by Nrf2 activation. In addition, sequence analysis of peptides more extensively acetylated in the absence of GCLM revealed an enrichment of cysteine residues immediately surrounding the site of acetylation; which suggests a potential specific regulation by decreased glutathione content. Pathway analysis revealed that some pathways exhibited both differential protein expression and lysine acetylation, revealing a coordinated response involving transcriptional and post-translational regulation.