Mucin-type O-glycosylation (MTOG) is commonly found on many of the cluster of differentiation (CD) antigens present on immune cells. Mammalian cells display long surface molecules (LSM) (e.g. CD43, CD45, and CD162) which act as shield and regulate cell-cell interactions. Particularly, CD43 (leukosialin / sialophorin) is estimated to carry 80-90 O-glycans on its polypeptide backbone and is considered to be a negative regulator of immune activation. The glycan load and molecular weight of CD43 depends on the specific cell type, cell state, and the cellular glycosylation machinery. CD43 contains 93 (46 Ser and 47 Thr) potential MTOG residues on its extracellular domain. Glycoproteomics based investigations of glycoproteins carrying high-occupancy clustered glycans using mass spectrometry has been a challenge due to protease resistance, limitations of low charge and high mass values, and poor fragmentation. We endeavoured to enumerate the diversity and complexity of glycopeptides from CD43 employing the high-energy collision dissociation – product dependent – electron transfer dissociation (HCD-PD-ETD) methodology. Extracellular domain of CD43 fused with Fc and His-tag (CD43ex-Fc-His) was stably expressed in Jurkat cells through lentiviral transduction and cultured in chemically defined medium conditions. CD43ex-Fc-His was purified from the supernatant, subjected to digestion by trypsin and Glu-C, and analyzed by nano-LC-ESI-MS/MS. The mass spectrometry data on glycopeptides were analyzed using Byonic software and manual validation, taking in to account all theoretical possibilities (e.g. unoccupied, HexNAc, NeuAcHexNAc, HexHexNAc, NeuAcHexHexNAc, and NeuAc2HexHexNAc). For example, allowing for only HexNAc, we detected 17/32 glycoforms for the undecapeptide 44-MYTTSITSDPK-54, which was reduced to 11/32 glycoforms upon treatment with Ac5GalNTGc, an efficient inhibitor of MTOG. Herein we will present results of our investigations and discuss the hypothesis that appropriate MTOG on CD43 is essential for its immune functions, both to act as a barrier as well as in regulating the later mobility on the plasma membrane.