Data explorer 4

An Applied Biosystems 4800 MALDI TOF/TOF^TM Analyser (Framingham, MA, USA) mass spectrometer was employed in this study to acquire MALDI and MS/MS spectra with and without using a collision gas (air or argon). This TOF/TOF instrument is equipped with a Nd:YAG laser with 355 nm wavelength, pulse of <500 ps, and 200 Hz repetition rate in both MS and MS/MS modes. The precursor ion selector of the analyser has a mass resolution of about 400. All measurements were performed in automatic mode. For MS/MS experiments, the potential difference between the source acceleration voltage and the collision cell was set at 2 kV. MALDI-MS e MALDI-TOF/TOF-MS/MS spectra were recorded in reflector positive ion mode. MS and MS/MS data were processed using Data Explorer 4.4 (Applied Biosystems, Framingham, MA, USA). Mass resolution (FWMH) was about 13,000 in MS mode and ranged from 1500 (m/z < 500) to 5000 (500 < m/z < 1300) in MS/MS mode.

Twenty micromoles of 2′-deoxyuridine 5′-monophosphate (dUMP), 1 mM CH₂O or ¹³C-labeled CH₂O, and 1 mM NADPH were incubated for 30 min in anaerobic conditions at room temperature with 50 µM of ThyX in 100 µL of 50 mM Tris pH 7.4 and 150 mM NaCl. The reaction was stopped by adding 100 µL of acidic phenol. Nucleotides in the aqueous phase were recovered after centrifugation and analyzed by high-performance liquid chromatography or NMR. Matrix-assisted laser desorption/ionization MS analyses were performed directly on the aqueous phase after phenol treatment of the samples using an UltrafleXtreme spectrometer (Bruker Daltonique, France). The instrument is equipped with an Nd:YAG laser (operating at 355 nm wavelength of <500 ps pulse and 200 Hz repetition rate). Acquisitions were performed in a negative ion mode. MS data were processed using DataExplorer 4.4 (Applied Biosystems).

The analysis of the permethylated N-glycan structures was performed employing matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF MS and MALDI-TOF/TOF MS/MS) mass spectrometry. MS and MS/MS data were acquired using a 4800 MALDI-TOF/TOF (Applied Biosystems) mass spectrometer. Permethylated samples were dissolved in 10 μl of methanol, and 1 μl of dissolved sample was premixed with 1 μl of matrix (10 mg/ml 3,4-diaminobenzophenone in 75% (v/v) aqueous acetonitrile), spotted onto a target plate, and dried under vacuum. For the MS/MS studies, the collision energy was set to 1 kV, and argon was used as collision gas. The 4700 Calibration standard kit, calmix (Applied Biosystems), was used as the external calibrant for the MS mode, and [Glu1] fibrinopeptide B human (Sigma) was used as an external calibrant for the MS/MS mode.
For analysis of mass spectra, data were processed using Data Explorer 4.9 Software (Applied Biosystems). The processed spectra were subjected to manual assignment and annotation with the aid of a glycobioinformatics tool, GlycoWorkBench [39 (link)]. The proposed assignments for the selected peaks were based on ¹²C isotopic composition together with knowledge of the biosynthetic pathways. Proposed structures were then confirmed by data obtained from MS/MS.

N-linked glycan analysis from RBC ghosts was performed according to Jang-Lee et al.^{46 (link)}. MS and MS/MS data from the permethylated purified glycan fractions were acquired on a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems). Data were processed using Data Explorer 4.9 Software (Applied Biosystems). The processed spectra were subjected to manual assignment and annotation with the aid of a glycobioinformatics tool, GlycoWorkBench.^{47 (link)} Proposed assignments for the selected peaks were based on ¹²C isotopic composition together with knowledge of the biosynthetic pathways, and structures were confirmed by data obtained from MS/MS experiments.

Glycomic profiles were acquired on a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer from Voyager-DE^TM STR PerSeptive Biosystems. Permethylated samples were dissolved in 10 μl of methanol from which 0.5 μl was added to 0.5 μl of the matrix (20 mg/ml of 2,5-dihydrobenzoic acid in 70% (v/v) aqueous methanol). The sample/matrix mixture was then loaded onto the sample plate for the mass spectrometric analysis. MALDI-TOF/TOF experiments were performed with a 4800 Proteomics Analyzer (Applied Biosystems) using 3,4-diaminobenzophenone (20 mg/ml in 70% (v/v) aqueous acetonitrile) as the matrix. Both instruments were operated in the reflectron positive mode. All MS and MS/MS data were processed using Data Explorer 4.9 software (Applied Biosystems).

MS and MS/MS data were acquired a 4800 MALDI-TOF/TOF (Applied Biosystems, Darmstadt, Germany) mass spectrometer. Permethylated glycans were dissolved in 10 μl of methanol, and 1 μl of dissolved sample was premixed with 1 μl of matrix (10 mg/ml 3,4-diaminobenzophenone (#184800250, Acros Organics) in 75% (v/v) aqueous acetonitrile), spotted onto a target plate, and dried under vacuum. For the MS/MS studies the collision energy was set to 1 kV, and argon was used as collision gas. The 4700 Calibration standard kit, calmix (Applied Biosystems), was used as the external calibrant for the MS mode, and [Glu1] fibrinopeptide B human (Sigma) was used as an external calibrant for the MS/MS mode.
The MS and MS/MS data were processed using Data Explorer 4.9 Software (Applied Biosystems). The processed spectra were subjected to manual assignment and annotation with the aid of a glycobioinformatics tool, GlycoWorkBench^{36 (link)}. The proposed assignments for the selected peaks were based on ¹²C isotopic composition together with knowledge of the biosynthetic pathways. The proposed structures were then confirmed by data obtained from MS/MS analysis. The bar chart graph was prepared with Originlab with data prepared in Microsoft Excel with relative abundances deriving from the Data Explorer software. All figures were prepared/finalised in Adobe Illustrator.