Mtp anchorchip 800 384 tf maldi target

MALDI-TOF-MS analysis of released N-glycans was performed as described previously (Selman et al., 2011 (link); Fischöder et al., 2019 (link)). Briefly, 0.9 cm cotton rope was used for Cotton HILIC SPE. The stationary phase was equilibrated with 50 µL LC-MS grade H₂O followed by 50 µL 85% ACN_aq. 10 µL of released N-glycans were adjusted to 70 µL 85% ACN_aq with 1% TFA and loaded onto the HILIC phase. Following two washing steps with 50 µL 85% ACN_aq with 1% TFA and 50 µL 85% ACN_aq, the samples were eluted in 70 µL LC-MS grade H₂O, vacuum evaporated and dissolved in 20 µL LC-MS grade H₂O. For the MALDI-TOF-MS analysis 0.5 µL super-dihydroxybenzoic acid (S-DHB) (≥99.0%, Sigma-Aldrich, Steinheim, Germany) matrix (10 mg/ml) in 30% (v/v) ACN_aq, 0.1% (v/v) TFA, 2 mM NaCl was spotted onto a MTP AnchorChip 800/384 TF MALDI target (Bruker Daltonics, Bremen, Germany). Subsequently 1 µL sample was applied onto the dried matrix layer. Measurements were carried out on an ultrafleXtreme MALDI-TOF/TOF MS (Bruker Daltonics, Bremen, Germany) in reflectron positive ion mode. Data was processed with the top-hat filter and the adjacent-averaging algorithm using flexAnalysis version 3.3 Build 80 (Bruker Daltonics, Bremen, Germany).

Each sample was measured once by mixing 5 μL of purified derivatized glycans with 0.5 μL of 5 mg/mL 2,5-dihydroxybenzoic acid (Bruker Daltonics, Bremen, Germany) 1 mM NaOH in 50 % ACN on a MTP AnchorChip 800/384 TF MALDI target (Bruker Daltonics) and left to dry at room temperature. Subsequently, the dried sample spots were recrystallized by adding 0.2 μL ethanol. All analyses were performed on an UltraFlextreme MALDI-TOF/TOF-MS equipped with a Smartbeam II laser, controlled by proprietary software Flexcontrol 3.4 (Bruker Daltonics). The Ultraflex was operated in reflectron positive (RP) ion mode, calibrated with a peptide calibration standard (Bruker Daltonics). For sample measurements 10,000 laser shots were accumulated at a laser frequency of 1000 Hz, using a complete sample random walk with 200 shots per raster spot. Tandem mass spectrometry (MALDI-TOF-MS/MS) was performed on the most abundant peaks of the mouse N-glycome via laser-induced dissociation. Fragmentation spectra were annotated using GlycoWorkbench (version 2.1) [32 ].