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Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow


Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.

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Fig. 1: Schematic of the PCMF setup.
Fig. 2: Comparison of the spray stability during elution when using nano-PGC-LC-ESI-MS(/MS) in negative ion mode.
Fig. 3: Comparison of the BPC of eluted N- and O-glycans released from bovine fetuin before (a) and after (b) PCMF supplementation.
Fig. 4: Comparison of the EIC of selected masses corresponding to N- and O-glycans released from bovine fetuin before (a) and after (b) PCMF supplementation.



ammonium bicarbonate


base peak chromatogram


octadecyl carbon chain




extracted ion chromatogram


electrospray ionization


higher-energy collisional dissociation


hydrophilic interaction liquid chromatography


high-performance liquid chromatography




inner diameter


potassium hydroxide


liquid chromatography coupled to mass spectrometry


mass spectrometry


tandem mass spectrometry

NaBH4 :

sodium borohydride


outer diameter


post-column make-up flow


porous graphitized carbon


ultra performance liquid chromatography


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TNK and ER were supported by the European Commission (EC) under the FP7 project “HTP-GlycoMet – Methods for high-throughput glycoproteomic analysis” (grant no. 324400). ER acknowledges support by the German Research Foundation (DFG) under the project “The concert of dolichol-based glycosylation: from molecules to disease models” (grant identifier FOR2509), by the German Federal Ministry of Education and Research (BMBF) under the project “Die Golgi Glykan Fabrik 2.0” (grant identifier 031C557), and by the European Commission (EC) under the H2020 project “IMforFuture” (grant no. 721815).

We want to thank Marcus Hoffmann and Alexander Behne for their contribution in editing the manuscript.

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Correspondence to Erdmann Rapp.

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Nguyen-Khuong, T., Pralow, A., Reichl, U. et al. Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow. Glycoconj J 35, 499–509 (2018). https://doi.org/10.1007/s10719-018-9848-1

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  • Glycans
  • Glycomics
  • Mass spectrometry
  • Nano-electrospray
  • Nano-liquid chromatography
  • Negative ion mode
  • Orbitrap
  • Porous graphitized carbon
  • Post-column make-up flow
  • Stability