FIGURE 4.

Determination of the polysialylated N-glycosylation site of ST8SiaII. A, purified polySia carriers of rat epididymis were separated by SDS-PAGE. Western blot (WB) analysis was performed using anti-polySia mAb 735 with or without previous endoN or PNGaseF treatment. Apparent molecular masses of standard proteins are indicated in kDa. B, MALDI-TOF mass spectra of precipitated polysialylated glycopeptides before (−) or after (+) PNGaseF treatment and nanoLC separation. Monoisotopic masses of the pseudomolecular ions [M + H]⁺ are given. C, fragmentation analysis of the de-N-glycosylated peptide by MALDI-TOF-MS/MS. Sequence-specific ions are labeled according to previous studies (44, 45). The conversion of Asn to Asp is illustrated in boldface. D, assignment of a polySia chain to individual N-glycosylation sites of ST8SiaII.