Frc 10a fraction collector

Assays against random glycopeptides GPIIC (Fig. 1b) (Sussex Research, Ottawa, CN) consisting of 100 μl reactions in 100 mM sodium cacodylic pH 6.5, 0.8 mM β-mercaptoethanol, 0.08% Triton X100, 10 mM Mn²⁺, ~ 0.8 mM GPIIC glycopeptide, 0.4 mM UDP-[³H]GalNAc, 1.4 μM TxgGalNAc-T3, were incubated at 37 °C on a shaking microincubator for 5 hr and 21 hr. Reactions were quenched with 100 μl of 250 mM EDTA, diluted to 4 ml and passed over a 1 ml column of DOWEX 1×8 (Sigma Aldrich). Dowex column flow through was lyophilized and applied to a Sephadex G10 column (Cytiva) to separate peptide from free [³H]GalNAc and the peptide peak was lyophilized for sequencing. Glycopeptide products were Edman sequenced on a modified gradient Shimadzu PPSQ53A sequencer (Shimadzu Scientific instruments Inc., Columbia, MD). The ³H-GalNAc-O-Ser-PTH derivatives (8.5-12 min) at each cycle were collected on a Shimadzu FRC-10A fraction collector and ³H-scintillation counted on a Beckman LS6500 scintillation counter.

For O-glycosylation analysis of glycopeptides CST1.2, CST1.3, CST1.4, SRS13.2, SRS13.4, Muc5AC-3, Muc5AC-3,13 and Muc5AC-13, (AnaSpec, Fremont, CA) by Edman Sequencing, 41-46 μl reactions consisting of 110-120 mM sodium cacodylic pH 6.5, 0.8 mM β-mercaptoethanol, 0.08% Triton X100, 12 mM Mn²⁺, 0.5-0.6 mM (glyco)peptides, 2.2-2.4 mM UDP-[³H]GalNAc, and 0.06-0.15 μM TxgGalNAc-T3 were incubated at 37 °C for 30 min (CST1.x) or 90 min (SRS13.x & Muc5ACx). Overnight incubations were also performed for CST1.3 and Muc5AC-13. Reactions were quenched with 100 μl 250 mM EDTA and processed as described for GPIIC. Pre and post Dowex ³H DPM and ³H DPM integration of the Sephadex G10 glycopeptide and GalNAc peaks were used to calculate percent of glycopeptide glycosylation and revealed no UDP-[³H]GalNAc hydrolysis against these glycopeptides. Glycopeptide products were Edman sequenced on a modified gradient Shimadzu PPSQ53A sequencer. Free GalNAc (2.25-3.25 min) and the GalNAc-O-Thr-PTH and GalNAc-O-Ser-PTH derivatives (9.0-14.5 min) at each cycle were collected on a Shimadzu FRC-10A fraction collector and each fraction scintillation counted on a Beckman LS6500 scintillation counter. Sequence chromatograms of the PTH (phenylthiohydantoin) derivatives were also analyzed as described in Supplemental Fig. S2j, k.