Journal
GLYCOCONJUGATE JOURNAL
Volume 23, Issue 5-6, Pages 355-369Publisher
SPRINGER
DOI: 10.1007/s10719-006-8492-3
Keywords
glycan sequencing; high energy CID MS; MS; MALDI TOF; TOF; mass spectrometry
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Concerted MALDI-MS profiling and CID MS/MS sequencing of permethylated glycans is one of the most effective approaches for high throughput glycomics applications. In essence, the identification of larger complex type N-glycans necessitates an unambiguous definition of any modification on the trimannosyl core and the complement of non-reducing terminal sequences which constitute the respective antennary structures. Permethylation not only affords analyses of both neutral and sialylated glycans at comparable ease and sensitivity but also yields more sequence-informative fragmentation pattern. Facile glycosidic cleavages directed mostly at N-acetylglucosamine under low energy CID, as implemented on a quadrupole/time-of-flight (Q/TOF) instrument, often afford multiple losses of the attached antenna resulting in characteristic ions related to the number of antennary branches on the trimannosyl core. Non-reducing terminal epitopes can be easily deduced but information on the linkage specific substituent on the terminal units is often missing. The high energy CID MS/MS afforded by TOF/TOF instrument can fill in the gap by giving an array of additional cross-ring and satellite ions. Glycosidic cleavages occurring specifically in concert with loss of 2-linked or 3-linked substituents provide an effective way to identify the branch-specific antennary extension. These characteristics are shown here to be effective in deriving the sequences of additionally galactosylated, sialylated and fucosylated terminal N-acetyllactosamine units and their antennary location. Together, a highly reproducible fragmentation pattern can be formulated to simplify spectral assignment. This work also provides first real examples of sequencing multiply sialylated complex type N-glycans by high energy CID on a TOF/TOF instrument.
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