Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans |
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Authors: | Shin-Yi Yu Sz-Wei Wu Kay-Hooi Khoo |
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Institution: | (1) Institute of Biological Chemistry, Academia Sinica, Taiwan;(2) National Core Facilities for Proteomics Research, Academia Sinica, Taiwan |
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Abstract: | 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.
Shin-Yi Yu and Sz-Wei Wu contributed equally to this work.
Dedicated to the late Prof. Yasuo Inoue, without whom the body of work represented by this article would not have been initiated
in Taiwan. |
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Keywords: | Glycan sequencing High energy CID MS/MS MALDI TOF/TOF Mass spectrometry |
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