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

Nature Protocols volume 6, pages 253–269 (2011)Cite this article

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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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Figure 1: A general overview of the procedure for profiling human plasma glycoproteomics.
Figure 2: Chromatograms for plasma immunodepletion and 2D HPLC chromatography.
Figure 3: Comparison of on-plate glycopeptide enrichment with two types of MALDI plates.
Figure 4: Distribution of crystallized GP-1 and GP-2 glycopeptides in a μFocus plate across the spotted area.
Figure 5: MALDI-DIT mass spectra of transferrin glycopeptides GP-1 and GP-2 at four different positions (outside, middle, inside, center) in the μFocus plate.
Figure 6: Data analysis overview.
Figure 7: Glycopeptide MS/MS ions search process.
Figure 8: Effect of collision energy on collision-induced dissociation MS/MS analysis of transferrin glycopeptides GP-1 and GP-2.
Figure 9: Deamination of a glycopeptide with N-terminal glutamine with MALDI-DIT collision-induced dissociation MS/MS.
Figure 10: Precursor selection for MS/MS analysis with MALDI-DIT.
Figure 11: Glycosylation heterogeneity at a single site in α-1-acid glycoprotein (encoded by ORM2).

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Author information

Authors and 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.

Corresponding author

Correspondence to Hong Wang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

Contains input files, MS1 spectra, MS2 spectra and analysis output for Well A10. (ZIP 23419 kb)

Supplementary Data 2

Contains input files, MS1 spectrum, MS2 spectra and analysis output for Well A12. (ZIP 16034 kb)

Supplementary Figure 1

Total ion chromatogram of transferrin tryptic digest analyzed by on-line nano RPLC-ESI-LTQFT. (PPT 87 kb)

Supplementary Figure 2

Isotopic peak distribution for the identified glycopeptide with the sequence R.NEEYN*K.S and the glycan composition 5Hex4HexNAc1Sia. (PPT 141 kb)

Supplementary Table 1

Efficiency of glycopeptides enrichment by hydrophilic interaction chromatography. (DOC 37 kb)

Supplementary Table 2

Protein analysis using nano LC-ESI LTQ-FT. (PDF 51 kb)

Supplementary Table 3

Analysis of glycoproteins in a human plasma fraction by LC MALDI-DIT MS/MS. (PDF 62 kb)

Supplementary Program

Program.zip (21MB) file contains programs, N-glycan database, protein database, refinement databases and readme. (ZIP 21409 kb)

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Wang, H., Wong, CH., Chin, A. et al. Integrated mass spectrometry–based analysis of plasma glycoproteins and their glycan modifications. Nat Protoc 6, 253–269 (2011). https://doi.org/10.1038/nprot.2010.176

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