skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry

Abstract

The N-glycan diversity of human serum glycoproteins, i.e. the human blood serum N-glycome, is complex due to the range of glycan structures potentially synthesizable by human glycosylation enzymes. The reported glycome, however, is limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report, several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to provide an improved view of glycan diversity. Sample preparation improvements include acidified, microwave-accelerated, PNGase F N-glycan release, and sodium borohydride reduction were optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased the sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. On-line separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient which provides additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) has been utilized. When method improvements aremore » combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described1 these technologies demonstrate the ability to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrate application of these advances in the context of the human serum glycome, and for which our initial observations include detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.« less

Authors:
; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1168903
Report Number(s):
PNNL-SA-102819
47418; KP1601010
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry, 86(17):8700-10
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Kronewitter, Scott R., Marginean, Ioan, Cox, Jonathan T., Zhao, Rui, Hagler, Clay D., Shukla, Anil K., Carlson, Timothy S., Adkins, Joshua N., Camp, David G., Moore, Ronald J., Rodland, Karin D., and Smith, Richard D. Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry. United States: N. p., 2014. Web. doi:10.1021/ac501839b.
Kronewitter, Scott R., Marginean, Ioan, Cox, Jonathan T., Zhao, Rui, Hagler, Clay D., Shukla, Anil K., Carlson, Timothy S., Adkins, Joshua N., Camp, David G., Moore, Ronald J., Rodland, Karin D., & Smith, Richard D. Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry. United States. doi:10.1021/ac501839b.
Kronewitter, Scott R., Marginean, Ioan, Cox, Jonathan T., Zhao, Rui, Hagler, Clay D., Shukla, Anil K., Carlson, Timothy S., Adkins, Joshua N., Camp, David G., Moore, Ronald J., Rodland, Karin D., and Smith, Richard D. Tue . "Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry". United States. doi:10.1021/ac501839b.
@article{osti_1168903,
title = {Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry},
author = {Kronewitter, Scott R. and Marginean, Ioan and Cox, Jonathan T. and Zhao, Rui and Hagler, Clay D. and Shukla, Anil K. and Carlson, Timothy S. and Adkins, Joshua N. and Camp, David G. and Moore, Ronald J. and Rodland, Karin D. and Smith, Richard D.},
abstractNote = {The N-glycan diversity of human serum glycoproteins, i.e. the human blood serum N-glycome, is complex due to the range of glycan structures potentially synthesizable by human glycosylation enzymes. The reported glycome, however, is limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report, several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to provide an improved view of glycan diversity. Sample preparation improvements include acidified, microwave-accelerated, PNGase F N-glycan release, and sodium borohydride reduction were optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased the sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. On-line separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient which provides additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) has been utilized. When method improvements are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described1 these technologies demonstrate the ability to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrate application of these advances in the context of the human serum glycome, and for which our initial observations include detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.},
doi = {10.1021/ac501839b},
journal = {Analytical Chemistry, 86(17):8700-10},
number = ,
volume = ,
place = {United States},
year = {Tue Sep 02 00:00:00 EDT 2014},
month = {Tue Sep 02 00:00:00 EDT 2014}
}