Fig. 1.

Outline of modified workflow. SimpleCell pellet is subjected to lysis, followed by protease and neuraminidase digestion; this is followed by O-glycopeptide enrichment by LWAC on a long column of immobilized VVA. In parallel, culture medium is treated with neuraminidase and then subjected to LWAC on a short column of VVA; this is followed by protease and neuraminidase digestion and LWAC enrichment on a long VVA column as for the cell lysate. After a pre-screening nLC-MS run to determine which LWAC fractions contained the most O-glycopeptides, these were divided into portions, with one part of each submitted directly to nLC-MS and the other parts pooled and submitted to IEF fractionation. IEF fractions (12) were then submitted to nLC-MS. Following computational processing of nLC-MS data as described, all spectral identifications were validated by inspection.