Protocol | Published:

Integrated mass spectrometry–based analysis of plasma glycoproteins and their glycan modifications

Nature Protocols volume 6, pages 253269 (2011) | Download Citation

Abstract

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography–multistage tandem mass spectrometry (LC-MS/MSn) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MSn analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.

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Author information

Affiliations

  1. Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • Hong Wang
    • , Chee-Hong Wong
    • , Alice Chin
    • , Ayumu Taguchi
    • , Allen Taylor
    •  & Samir Hanash
  2. Ventana Medical Systems, Oro Valley, Arizona, USA.

    • Hong Wang
  3. Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan.

    • Sadanori Sekiya
    • , Hidenori Takahashi
    • , Masaki Murase
    • , Shinichi Iwamoto
    •  & Koichi Tanaka
  4. Technology Research Laboratory, Shimadzu Corporation, Seika-cho, Soraku-gun, Kyoto, Japan.

    • Shigeki Kajihara

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Contributions

H.W., K.T. and S.H. conceived and supervised the project. H.W. contributed to the development and evaluation of the methods and combined the data and wrote the protocol. C.-H.W. designed and developed the analysis software. A.C. performed the sample fractionation. H.W. and S.S. contributed to the mass spectrometry analysis of the sample. H.T. and S.I. optimized the MALDI-DIT instrument. A. Taguchi, A. Taylor and A.C. prepared the analyzed sample. H.T., M.M. and S.K. designed and developed the MALDI-DIT software.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hong Wang.

Supplementary information

Zip files

  1. 1.

    Supplementary Data 1

    Contains input files, MS1 spectra, MS2 spectra and analysis output for Well A10.

  2. 2.

    Supplementary Data 2

    Contains input files, MS1 spectrum, MS2 spectra and analysis output for Well A12.

  3. 3.

    Supplementary Program

    Program.zip (21MB) file contains programs, N-glycan database, protein database, refinement databases and readme.

Powerpoint files

  1. 1.

    Supplementary Figure 1

    Total ion chromatogram of transferrin tryptic digest analyzed by on-line nano RPLC-ESI-LTQFT.

  2. 2.

    Supplementary Figure 2

    Isotopic peak distribution for the identified glycopeptide with the sequence R.NEEYN*K.S and the glycan composition 5Hex4HexNAc1Sia.

Word documents

  1. 1.

    Supplementary Table 1

    Efficiency of glycopeptides enrichment by hydrophilic interaction chromatography.

PDF files

  1. 1.

    Supplementary Table 2

    Protein analysis using nano LC-ESI LTQ-FT.

  2. 2.

    Supplementary Table 3

    Analysis of glycoproteins in a human plasma fraction by LC MALDI-DIT MS/MS.

About this article

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DOI

https://doi.org/10.1038/nprot.2010.176

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