Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans.
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.
Yu
Shin-Yi
SY
Institute of Biological Chemistry, Academia Sinica, Taiwan.
Wu
Sz-Wei
SW
Khoo
Kay-Hooi
KH
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D006224:Cricetinae; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D015027:Zebrafish
D011134:Polysaccharides; D009584:Nitrogen