Post-column make-up flow (PCMF) enhances the performance of capillary-flow PGC-LC-MS/MS-based glycomics

Hannes Hinneburg, Sayantani Chatterjee, Falko Schirmeister, Terry Nguyen-Khuong, Nicolle H. Packer, Erdmann Rapp, Morten Thaysen-Andersen

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon–liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc.2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.
LanguageEnglish
Pages4559-4567
Number of pages9
JournalAnalytical Chemistry
Volume91
Issue number7
DOIs
Publication statusPublished - 2 Apr 2019

Fingerprint

Capillary flow
Polysaccharides
2-Propanol
Organic solvents
Glycoproteins
Glycomics
Ions
Liquid chromatography
Ion sources
Isomers
Mass spectrometry
Methanol
Signal to noise ratio
Carbon

Cite this

@article{6e4efa6af73646b49cb3afacb9a16345,
title = "Post-column make-up flow (PCMF) enhances the performance of capillary-flow PGC-LC-MS/MS-based glycomics",
abstract = "Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon–liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc.2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100{\%} ACN and IPA (equating to 57{\%} (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.",
author = "Hannes Hinneburg and Sayantani Chatterjee and Falko Schirmeister and Terry Nguyen-Khuong and Packer, {Nicolle H.} and Erdmann Rapp and Morten Thaysen-Andersen",
year = "2019",
month = "4",
day = "2",
doi = "10.1021/acs.analchem.8b05720",
language = "English",
volume = "91",
pages = "4559--4567",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "7",

}

Post-column make-up flow (PCMF) enhances the performance of capillary-flow PGC-LC-MS/MS-based glycomics. / Hinneburg, Hannes; Chatterjee, Sayantani; Schirmeister, Falko; Nguyen-Khuong, Terry; Packer, Nicolle H.; Rapp, Erdmann; Thaysen-Andersen, Morten.

In: Analytical Chemistry, Vol. 91, No. 7, 02.04.2019, p. 4559-4567.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Post-column make-up flow (PCMF) enhances the performance of capillary-flow PGC-LC-MS/MS-based glycomics

AU - Hinneburg, Hannes

AU - Chatterjee, Sayantani

AU - Schirmeister, Falko

AU - Nguyen-Khuong, Terry

AU - Packer, Nicolle H.

AU - Rapp, Erdmann

AU - Thaysen-Andersen, Morten

PY - 2019/4/2

Y1 - 2019/4/2

N2 - Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon–liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc.2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.

AB - Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon–liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc.2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.

UR - http://www.scopus.com/inward/record.url?scp=85063132410&partnerID=8YFLogxK

U2 - 10.1021/acs.analchem.8b05720

DO - 10.1021/acs.analchem.8b05720

M3 - Article

VL - 91

SP - 4559

EP - 4567

JO - Analytical Chemistry

T2 - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 7

ER -