AbstractThe membrane protein CD9P‐1 is a major component of the tetraspanin web, a network of molecular interactions in the plasma membrane, in which it specifically associates with tetraspanins CD9 and CD81. The various functional effects of CD9 and CD81 may be related to their partners. Thus, we have addressed the characterization of the CD9P‐1 glycosylation using stably transfected HEK‐293 cells. After immunoprecipitation, CD9P‐1 was subjected to enzymatic PNGase F cleavage of N‐glycans, resulting in Asn to Asp conversion and increase in 1 mass unit. Thus, following protease digestion, deglycosylated peptides were selectively identified by high mass accuracy FTICR‐MS, using this conversion as a signature. This has demonstrated that all nine potential N‐glycosylation sites were actually engaged. On the other hand, the N‐glycan structures were determined combining chemical derivatization and exoglycosidase digestions followed by MALDI‐TOF MS, ESI‐MS/MS, and GC‐MS analysis. CD9P‐1 was shown to exhibit more than 40 different N‐glycans, essentially composed of complex and high mannose‐type structures. Finally, 2‐D PAGE and lectino‐blot analyses have revealed the presence of at least 17 glycosylated isoforms of CD9P‐1 at cell surface. All CD9P‐1 isoforms associate with CD9 leading to additional level of complexity of this primary complex in the tetraspanin web.