Updated project metadata. Cancer-derived small extracellular vesicles have been proposed as promising potential biomarkers for diagnosis and prognosis of breast cancer (BC). We performed a proteomic study of lysine acetylation of breast cancer-derived small extracellular vesicles (sEVs) to understand the potential role of the aberrant acetylated proteins in the metastasis and progression of BC. Three cell lines were used as models for this study: MCF10A (non-metastatic), MCF7 (estrogen and progesterone receptor positive, metastatic) and MDA-MB-231 (triple-negative, highly metastatic). For a com-prehensive protein acetylation analysis of the sEVs derived from each cell line, acetylated peptides were enriched using the anti-acetyl-lysine antibody, followed by LC-MS/MS analysis. In total, 118 lysine acetylated peptides of which 22, 58 and 82 have been identified in MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These acetylated peptides were mapped to 60 distinct pro-teins and mainly identified proteins involved in metabolic pathways. Among those identified acetylated proteins presented in cancer-derived sEVs (MCF7, MDA-MB-231) are proteins associ-ated with glycolysis pathway, annexins and histones. Five enzymes from the glycolytic pathway, present only in sEVs isolated from cancer-derived sEVs cell lines, were validated. These include aldolase (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK 1), enolase (ENO) and pyruvate kinase M1/2 (PKM). For three of these enzymes, ALDOA, PGK 1 and ENO, the specific enzymatic activity was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study reveals that sEVs contain acetylated glycolytic metabolic enzymes that could be interesting potential candidates for early BC diagnostics.