Ting Song
Ting Song; Danielle Aldredge; Javier González; Carlito Lebrilla
University of California Davis, Davis, California
NOVEL ASPECT: We constructed a serum-based library containing 164 structures with relative abundances, retention times, accurate masses, and tandem MS spectra.
Glycosylation is an abundant and common posttranslational modification of proteins with functions related to stabilization of protein conformation, secretion through membranes, protein turnover control, proteinase hydrolysis protection, increase of the protein solubility, and cell recognition. They are also an important new target for biomarker development. Here we present the construction of a serum-based N-glycan library with serial digestion using exoglycosidases monitored by nanoLC MS. The library will be the core of a method that employs nanoLC MS with accurate mass, reproducible retention times, and tandem MS spectra for the rapid throughput glycomics.
Human pooled serum N-glycans were released using PNGase-F digestion and chemically reduced with NaBH4. Released N-glycans were fractionated off-line by HPLC using a Hypercarb PGC column, and fractions were screened for glycan compositions using MALDI-FTICR-MS. Serial exoglycosidase treatments, which were monitored using nanoLC-Chip-Q-TOF-MS, allowed determination of N-glycan structures and linkages.
In a typical serum N-glycan nanoLC MS profile, more than 300 compounds are observed over five orders of magnitudein abundances. We have now constructed a library containing 164 N-glycan structures, of which 28 are fully elucidated, more than 80 partially elucidated, and the remainder are putative structures. Included in the library are the intensities in pooled human serum, retention time, accurate mass and fragmentation profile. Previously, our group developed an N-glycan database that was based on commercially available standards of the five most abundant glycoproteins in serum.  We expand on this work by annotating glycans released directly from serum.We created a systematic name for the N-glycans. For example, N5402a, where “N” means N-glycans, and the compositions for Hex:HexNAc:Fuc:NeuAc, followed a letter according to their relative abundances in serum, “a” being the most abundant. The two most abundant N-glycans in serum are isomers of the biantennary disialylated structure (N5402a and N5402b), where the structure of highest abundance has the sialic acid (NeuAc) linked α2-3 to the 1-6-antenna and α2-6 to the 1-3-antenna, while the structure of second highest abundance has the NeuAc residues on both antenna linked α2-3. Similarly two isomeric structures of the monosialylated N-glycan (N5401a and N5401b) could be resolved. Both structures contained NeuAc with α2-6 linkages, but N5401a has the NeuAc on the 1-3-antenna, while N5401b has the NeuAc on the 1-6-antenna.In conclusion, we constructed a serum-based library containing 164 structures with identifiers, relative abundances, retention times, accurate masses, and tandem MS spectra that will be used for the rapid throughput glycomics and for biomarker discovery with glycan structures.