This project aims to investigate the comprehensive post-translational modification (PTM) landscape of extracellular matrix (ECM) proteins, specifically focusing on the functional impact of PLOD3 extracellular activity on collagen lysyl hydroxylation and glycosylation. Conditioned medium (CM) containing secretomes was harvested from HEK-293T cells under three distinct genetic conditions: Control (Ctrl), PLOD3 Overexpression (Plod3 OE), and a dysfunctional PLOD3 mutant [Plod3 Dut (Y117A, D672A)]. Murine brain microvascular endothelial cells (bEnd.3) were treated with these respective CM variations for 72 hours. Subsequently, the cells were decellularized, and the remaining ECM scaffold was harvested. Following SDS-PAGE separation and Coomassie Brilliant Blue staining, the bands corresponding to Collagen Type XVIII (COL18) were meticulously excised and subjected to in-gel tryptic digestion. The digested peptide mixtures were analyzed via high-resolution LC-MS/MS. Protein identification, label-free quantification (LFQ), and site-specific K-residue modification profiling (including mono- and di-saccharide glycosylation) were fully characterized using Proteome Discoverer 2.