Smartbeam 2 laser

Derivatised glycan samples were measured using the reflectron positive ion mode of an Ultraflextreme MALDI-TOF-MS (Bruker Daltonics), equipped with a Smartbeam-II laser, and operated by flexControl 3.4 build 135. Prior to the measurement, external calibration was performed using a peptide calibration standard. Ions were accelerated at 25 kV with 140 ns delayed extraction. Per sum spectrum, a total of 20,000 shots were accumulated between m/z 1,000 and m/z 5,000.

An ImagePrep spray station (Bruker Daltonics, Billerica, MA, USA) was used to coat the slide with a 0.2 mL aqueous solution of PNGaseF (20 µg total/slide) as previously described [21 (link)]. As negative control, adjacent control tissue slices were shielded from PNGaseF application by covering the tissue section with a glass slide. Digestion occurred in a humidified chamber at 37 °C for 2 h. Slides were desiccated prior to α-cyano-4-hydroxycinnamic acid matrix application (0.021 g CHCA in 3 mL 50% acetonitrile/50% water and 12 µL 25% TFA) using the ImagePrep sprayer. Released glycan ions were detected using a Solarix dual source 7T FTICR mass spectrometer (Bruker Daltonics) (m/z 690–5000) with a SmartBeam II laser operating at 1000 Hz, a laser spot size of 25 μm. Following MS analysis, data was loaded into FlexImaging Software focusing on the range m/z = 1000–4000 and reduced to 0.95 ICR Reduction Noise Threshold. FlexImaging 4.0 (Bruker Daltonics) was used to generate images of differentially expressed glycans. Observed glycans were searched against the glycan database generated using GlycoWorkbench [25 (link)]. Presented glycan structures were generated in GlycoWorkbench and represent putative structures determined by combinations of accurate m/z and off-slide derivatization experiments. CASI/CID was done as previously described [20 (link),21 (link)].

From each purified sample, 10 µL was premixed with 10 µL matrix consisting of 5 mg/mL super-DHB in 99% ACN with 1 mM NaOH. Of this mixture, 2 µL was spotted onto a MALDI target plate (800/384 MTP AnchorChip, Bruker Daltonics, Bremen, Germany) and was left to dry. MALDI-TOF-MS spectra were recorded on an UltrafleXtreme mass spectrometer with a Smartbeam-II laser (Bruker Daltonics) in reflectron positive mode, controlled by flexControl 3.4 (Build 135). Measurements were performed within a range from m/z 1,000 to 5,000, accumulating 10,000 laser shots at a frequency of 1,000 Hz and with 100 shots per raster spot using a random walking pattern.

All samples were analyzed at two MALDI-TOF facilities (the University of Burgundy’s CLIPP facility (Dijon, France) and Lille University Hospital’s neonatal screening laboratory (Lille, France)), according to the previously described procedure [11 (link)]. At both facilities, samples were prepared for MS measurements using a research version of the NeoSickle^® kit (Biomaneo). Samples were deposited in quadruplicate on a 384-spot polished steel MALDI target (Bruker Daltonik GmbH). At both facilities, MS was performed with a MALDI-TOF system: An AutoFlex™ Speed with a 2000 Hz Smartbeam™ II laser (Bruker Daltonik GmbH) in Dijon, and an AutoFlex™ III with a 200 Hz Smartbeam^™ laser (Bruker Daltonik GmbH) in Lille.

Assays were performed using the same basic mixture as described above, except that 1 mM of an adapted form of the EA2 peptide with an C-terminal arginine rather than lysine residue (PTTDSTTPAPTTR) was employed. The products were analysed using a Bruker Autoflex Speed MALDI ToF/ToF mass spectrometer (equipped with a Smartbeam™-II laser) in positive mode; the samples were first dried under vacuum on a steel plate and then 6-aza-2-thiothymine (ATT) was applied twice as matrix. Using the LIFT mode, fragmentation of parent ions was performed by laser-induced dissociation and 4000 shots were sampled. MS/MS spectra were processed with the manufacturer's software (Bruker Flexanalysis 3.3.80) using the SNAP algorithm with a signal/noise threshold of 3 (four-times smoothed).