MALDI TOF MS and MS/MS analysis
A few microliters of permethylated N-glycan samples, resuspended in
MeOH, were mixed with the same volume of matrix solution (10 mg/ml
5-Chloro-2-mercaptobenzothiazole, CMBT in MeOH/H2O
80/20). MALDI TOF and MALDI TOF/TOF mass spectra were recorded in
reflectron positive mode using a 4800 MALDI TOF/TOF™ (Applied
Biosystems) instrument, equipped with an Nd:YAG laser at 355-nm and 200
Hz repetition rate. In MS mode, 1200 shots were accumulated for each
spectrum, with a resolution greater than 15K and a mass accuracy better
than 80 ppm. A 4700-calibration standard kit, calmix (Applied
Biosystems) was used as external calibrant for the MS mode, and
[Glu1] fibrinopeptide B human (Sigma) was used as external calibrant
for the MS/MS mode (1 μL of TOF/TOF Calibration Mixture in 24 μL of CHCA
matrix solution).
The MS data were processed by DataExplorer™ 4.9 software and the
N-glycan species were identified by bioinformatics tools, such as
GlycoMod (http://web.expasy.org/glycomod/), Glycoworkbench v2.1 [31]
and by tools provided by the Consortium for Functional Glycomics (CFG).