Mtp anchorchip 800 384 tf

MALDI-TOF(/TOF)-MS(/MS) analysis was performed on an UltrafleXtreme (Bruker Daltonics) operated under flexControl 3.3 (Build 108; Bruker Daltonics). One microliter of the enriched ethyl-esterified glycans was spotted on a MALDI target (MTP AnchorChip 800/384 TF; Bruker Daltonics) together with 1 μL 5 mg/mL super-DHB in 50% ACN and 1 mM NaOH. The spots were dried by air at room temperature. For each spot, a mass spectrum was recorded from m/z 1,000 to 3,000, combining 10,000 shots in a random walk pattern at 1,000 Hz and 100 shots per raster spot. Prior to the analysis of the samples, the instrument was calibrated using peptide calibration standard (Bruker Daltonics). MALDI-TOF/TOF-MS/MS of the most abundant peaks was performed by laser-induced dissociation.

The N-glycosylation of total plasma glycoproteins as well as affinity-purified IgG (75% of eluate) was analyzed by MALDI-TOF/TOF-MS/MS at the released N-glycan level. After enzymatic N-glycan release, sialic acids were stabilized by linkage-specific ethyl esterification [64 (link),65 (link)]. Briefly, released glycans were added to derivatization reagent and incubated for 1 h at 37 °C. The derivatized glycans were enriched by cotton hydrophilic-interaction liquid chromatography (HILIC)−solid-phase extraction (SPE) as described before and eluted in 10 μL water [64 (link),66 (link)]. MALDI-TOF(/TOF)-MS(/MS) analysis was performed on an UltrafleXtreme (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam-II laser. The enriched ethyl-esterified glycans (2 µL) were spotted on a MALDI target (MTP AnchorChip 800/384 TF; Bruker Daltonics, Bremen, Germany) together with 1 µL 5 mg/mL super-DHB in 50% ACN and 1 mM NaOH. The spots were dried by air at room temperature. Spectra were acquired with accumulation of 10.000 laser shots at a laser frequency of 1000 Hz, using a complete sample random walk with 200 shots per raster spot. Selected peaks were fragmented via laser-induced dissociation (MALDI-TOF/TOF-MS/MS).

MALDI-TOF-MS analysis was performed on an UltrafleXtreme (Bruker Daltonics) operated under flexControl 3.3 (Build 108; Bruker Daltonics). Two and 5 μL of the enriched ethyl esterified glycans were spotted on a MALDI target (MTP AnchorChip 800/384 TF; Bruker Daltonics) together with 1 μL of super-DHB (5 mg/mL in 50% ACN and 1 mM NaOH). The spots were dried by air at room temperature. For each spot, a mass spectrum was recorded in the range from m/z 1,000 to 5,000, combining 10,000 shots in a random walk pattern at 1,000 Hz and 200 shots per raster spot. Prior to the analysis of the samples, the instrument was calibrated using a peptide calibration standard (Bruker Daltonics). Tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) was performed for the most abundant glycans using laser-induced dissociation, and compositions as well as structural features of N-glycans were assessed on the basis of the observed fragment ions.