Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

Christopher Ashwood, Brian Pratt, Brendan X. Maclean, Rebekah L. Gundry, Nicolle H. Packer

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3601-3612
Number of pages12
JournalAnalyst
Volume144
Issue number11
DOIs
StatePublished - Jun 7 2019

Fingerprint

Glycomics
standardization
Standardization
Polysaccharides
Carbon
carbon
Dextran
Glucose
glucose
Libraries
Glycoproteins
Dextrans
Ladders
Chromatography
automation
Cell culture
Isomers
chromatography
Automation
Tissue

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy
  • Electrochemistry

Cite this

Ashwood, C., Pratt, B., Maclean, B. X., Gundry, R. L., & Packer, N. H. (2019). Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping. Analyst, 144(11), 3601-3612. https://doi.org/10.1039/c9an00486f

Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping. / Ashwood, Christopher; Pratt, Brian; Maclean, Brendan X.; Gundry, Rebekah L.; Packer, Nicolle H.

In: Analyst, Vol. 144, No. 11, 07.06.2019, p. 3601-3612.

Research output: Contribution to journalArticle

Ashwood, C, Pratt, B, Maclean, BX, Gundry, RL & Packer, NH 2019, 'Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping', Analyst, vol. 144, no. 11, pp. 3601-3612. https://doi.org/10.1039/c9an00486f
Ashwood, Christopher ; Pratt, Brian ; Maclean, Brendan X. ; Gundry, Rebekah L. ; Packer, Nicolle H. / Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping. In: Analyst. 2019 ; Vol. 144, No. 11. pp. 3601-3612.
@article{376d6f0f12ea480dbcc9d64b2fcb29e4,
title = "Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping",
abstract = "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.",
author = "Christopher Ashwood and Brian Pratt and Maclean, {Brendan X.} and Gundry, {Rebekah L.} and Packer, {Nicolle H.}",
year = "2019",
month = "6",
day = "7",
doi = "10.1039/c9an00486f",
language = "English (US)",
volume = "144",
pages = "3601--3612",
journal = "The Analyst",
issn = "0003-2654",
publisher = "Royal Society of Chemistry",
number = "11",

}

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.

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.

UR - http://www.scopus.com/inward/record.url?scp=85066401383&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85066401383&partnerID=8YFLogxK

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 -