Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7612266 | Journal of Chromatography A | 2014 | 34 Pages |
Abstract
We reported an improved combinatorial approach for identifying site-specific O-glycosylation using both glycan cleaved and non-cleaved methods. In this approach, a non-reducing β-elimination kit coupled with non-specific enzymes performed efficient digestion, O-glycan cleavage, and partial dephosphorylation without significant side reactions, thus enabling an automatic database search for the cleaved O-glycosylation or serine/threonine (S/T) phosphorylation sites. From the same sample concurrently prepared without β-elimination, the corresponding intact O-glycopeptides were mapped by accurate precursor ion mass using an in-house glycan database majorly composed of GalNAc (mucin-type) core and the retention-time shift (ÎRt). Each glycopeptide assignment was verified by the detection of glycan-specific fragments using collision-induced dissociation (CID) to estimate False Discovery Rate (FDR). Using fetuin as a model, all identified S/T elimination sites were matched to multiple intact glycopeptides with a 31% FDR. This considerably reduced to 0% FDR by ÎRt filtering. This approach was then applied to a protein mixture composed of therapeutic Factor IX and Enbrel® mixed with fetuin and kappa-casein. A total of 26 glycosylation sites each of which corresponds to 1-4 glycans were positively mapped and confirmed. The FDR decreased from 33% to 3.3% by ÎRt filtering and exclusion of repeated peptide tags that covered the same glycosylation sites. Moreover, the phosphorylation and O-glycosylation on the same site such as T159 of Factor IX and T170 of kappa-casein were able to be unambiguously differentiated. Thus, our approach is useful for in-depth characterization of site-specific O-glycosylation of a simple mixture such as protein-based therapeutics.
Keywords
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Li-Juan Huang, Jen-Hui Lin, Jung-Heng Tsai, Yen-Yin Chu, Yen-Wen Chen, Shun-Li Chen, Shu-Hui Chen,