In this study, we expand on this major advancement in O-GlcNAc enrichment protocols by adapting this approach to human-banked samples, which often presents their significant challenges. Indeed, patient samples are often banked without proper washing, thus contaminating cells with abundant O-glycosylated blood protein. These glycoproteins, such as human serum albumin, antibodies, and hemoglobin, pollute MS acquisition, masking the less abundant intracellular proteins and, even more, their O-GlcNAcylated forms. Thus, using freshly banked placentas, we present an adapted method to prepare human placental samples with naturally high blood content (13). We then enriched for O-GlcNAcylated protein using the PTMscan antibodies previously mentioned (12). However, our adapted protocol yielded over 60% of HexNAc peptide on total peptides. Per sample, we obtained up to 2,600 HexNAc-containing PSMs, 1,090 unique HexNAc peptides, and more than 1,000 unique HexNAc proteins using 45mg of starting placenta. Furthermore, we present a bioinformatic workflow to sort these HexNAc PSMs and extract the most confident O-GlcNAcylated proteins and sites. Thus, we confidentially identified 641 O-GlcNAcylated proteins, including up to 146 new proteins and 730 new sites within the 2 placental samples analyzed. Furthermore, we enriched for over 50 placenta-enriched O-GlcNAcylated proteins, such as RPA4, representing a new biomarker of the placental O-GlcNAcylation.