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Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MSn switching system

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Abstract

Four types of neutral glycosphingolipids (LacCer, Gb3Cer, Gb4Cer, and IV3αGalNAc-Gb4Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSn switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS2, and MS3 spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]+ in the positive ion mode mass spectra and [M−H]− in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS2 spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS3 mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS2, and MS3. The established HPLC-ESI-QIT-TOF MS with MSn switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb4Cer in a crude mixture prepared from human erythrocytes.

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Abbreviations

GlcCer:

Glucosylceramide

LacCer:

Lactosylceramide

Gb3Cer:

Globotriaosylceramide

Gb4Cer:

Globotetraosylceramide

IV3αGalNAc-Gb4Cer:

Forssman antigen

GSLs:

Glycosphingolipids

HPLC:

High-performance liquid chromatography

ESI:

Electrosprayionization

QIT-TOF:

Quadrupole ion trap time-of-flight

MS:

Mass spectrometry

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Acknowledgments

This work was supported by a grant for Supporting Research Centers in Private Universities.

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Authors and Affiliations

  1. Global Research Cluster, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan

    Emi Ito

  2. Analytical Division, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto, 604-8511, Japan

    Hiroaki Waki & Kozo Miseki

  3. Life Science Research Center, Shimadzu Corporation, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 105-0045, Japan

    Takashi Shimada, Taka-Aki Sato & Minoru Suzuki

  4. School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan

    Kazuaki Kakehi

  5. Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan

    Akemi Suzuki

Authors
  1. Emi Ito
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  2. Hiroaki Waki
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  3. Kozo Miseki
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  4. Takashi Shimada
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  6. Kazuaki Kakehi
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  7. Minoru Suzuki
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  8. Akemi Suzuki
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Correspondence to Akemi Suzuki.

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Ito, E., Waki, H., Miseki, K. et al. Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MSn switching system. Glycoconj J 30, 881–888 (2013). https://doi.org/10.1007/s10719-013-9492-8

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  • DOI: https://doi.org/10.1007/s10719-013-9492-8

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