TY - JOUR
T1 - Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping
AU - Ashwood, Christopher
AU - Pratt, Brian
AU - Maclean, Brendan X.
AU - Gundry, Rebekah L.
AU - Packer, Nicolle H.
N1 - Funding Information:
This work was supported by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003). Portions of this research were supported by grants from the National Institute of General Medical Sciences (R01 GM103551) and National Heart Lung and Blood Institute (R01 HL134010). We thank M. R. Hutchinson and V. Staikopoulos for providing mouse tissue sections.
PY - 2019/6/7
Y1 - 2019/6/7
N2 - 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 N-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.
AB - 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 N-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.
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U2 - 10.1039/c9an00486f
DO - 10.1039/c9an00486f
M3 - Article
C2 - 31065629
AN - SCOPUS:85066401383
VL - 144
SP - 3601
EP - 3612
JO - The Analyst
JF - The Analyst
SN - 0003-2654
IS - 11
ER -