<p></p><p>Porous graphitized carbon (PGC) based chromatography
achieves high-resolution separation of glycan structures released from
glycoproteins. This approach is especially valuable when resolving structurally
similar isomers and for discovery of novel and/or sample-specific glycan
structures. However, the implementation of PGC-based separations in glycomics
studies has been limited because system-independent retention values have not
been established to normalize technical variation. To address this limitation,
this study combined the use of hydrolyzed dextran as an internal standard and
Skyline software for post-acquisition normalization to reduce retention time and
peak area technical variation in PGC-based glycan analyses. This approach
allowed assignment of system-independent retention values that are applicable
to typical PGC-based glycan separations and supported the construction of a library
containing >300 PGC-separated glycan structures with normalized glucose unit
(GU) retention values. To enable the automation of this normalization method, a
spectral MS/MS library was developed of the dextran ladder, achieving
confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two
ways. First, to inform the search space for bioinformatically predicted but unobserved
glycan structures, predictive models for two structural modifications,
core-fucosylation and bisecting GlcNAc, were developed based on the GU library.
Second, the applicability of this method for the analysis of complex biological
samples is evidenced by the ability to discriminate between cell culture and
tissue sample types by the normalized intensity of <i>N-</i>glycan structures alone. Overall, the methods and data described
here are expected to support the future development of more automated approaches
to glycan identification and quantitation.</p><br><p></p>
Use of Principal Component Analysis for the Study of the Influence of Physicochemical Parameters on the Retention of Barbiturates on Porous Graphitized Carbon Column.
