Fibrinopeptide b human

A MALDI-TOF/TOF (AB SCIEX TOF/TOF 5800, Applied Biosystems, Framingham, MA) was used in this study for both MS and MS/MS analyses of glycosphingolipids. Extracted lipids were reconstituted in 100 μL acetonitrile–methanol (9:1, v/v) and approximately 0.5 μL was spotted on an AB SCIEX Opti-TOF MALDI plate. Sample spots were overlaid with 0.5 μL of the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) in an acetonitrile–methanol solution (5 mg/mL DHB in 9:1 ACN-MeOH, v/v). A 355 nm laser at a repetition rate of 200 Hz was employed for ionization. For MS analysis, mass spectra were the sum of 4000 laser shots and acquired in reflector positive mode. For MS/MS analysis, mass spectra were the sum of 1000 laser shots with collision energy of 2 keV and pressurized air was utilized as the collision gas to induce fragmentation. The following standards were used to calibrate the mass spectrometer: polypeptide hormones (ACTH I-III, Sigma–Aldrich, St. Louis, MO) and a peptide (Fibrinopeptide B, human, Sigma–Aldrich, St. Louis, MO). External GSL standards were spotted separately and used for the comparative analysis and quantification.

MS data were acquired by using either a Voyager DE-STRTM MALDI-TOF or a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Darmstadt, Germany). MS/MS data were acquired with the latter instrument. MS mode was calibrated with 4700 Calibration standard kit (Applied Biosystems), and MS/MS mode was calibrated with fibrinopeptide B human (Sigma). For MS/MS studies, the collision energy was set to 1 kV, and the collision gas was argon. 2, 5-dihydroxybenzoic acid was used as matrix. Permethylated samples were dissolved in 10 μL methanol, 1 μL of this solution was premixed with 1 μL matrix and1 μL of the mixture was spotted onto the plate.