Super dhb matrix

N-glycan analysis of the EPO reporter expressed from HEK293^WT treated with NSC80997 or DMSO was performed as described previously^{100 (link)}. Briefly, 10 µg purified EPO reporter was dried, resuspended in 10 µl 1% SDS in PBS and incubated at 60 °C for 30 min. Then 10 µl 2% NP-40/PBS containing 0.5 U PNGaseF (Roche) was added, and samples were incubated at 37°C overnight. The ethyl esterification derivatization was performed by adding 100 μL 0.25 M EDC/0.25 M HOBt in ethanol to the released glycan sample and incubating the mixture for 1 h at 37 °C. Cotton HILIC microtip purification was performed to obtain the purified and enriched N-glycans, and 3 µl of the mixture was spotted together with 1 µl super-DHB matrix (Sigma-Aldrich) onto a target steel plate. MALDI-TOF-MS spectra were recorded in reflectron positive mode on a Bruker Autoflex instrument (Bruker Daltonics) Spectra were recorded with an m/z-range of 1000–5000 Da.

Five microliter of eluate was mixed on the spot with sodium hydroxide-spiked Super-DHB matrix (Sigma-Aldrich, St. Louis, MO, USA) on an AnchorChip plate. Automated matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) measurements were performed summing 10,000 shots per spot with 250 shot steps and a full spot random walk. Details on data processing, extraction, and the calculation of glycosylation traits are described in Additional file 1.