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Mass spectrometry–based detection and quantification of plasma glycoproteins using selective reaction monitoring

Nature Protocols volume 7, pages 859–871 (2012)Cite this article

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Abstract

Mass spectrometry–based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.

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Figure 1: Chemical schematic of the glyco-capture process with an example of a terminal galactose.
Figure 2: Workflow of targeted quantification of N-glycopeptides in serum samples using the LC-SRM assay.
Figure 3: Example of glycoproteins (with known plasma concentration) identified in a typical LC-MS/MS run.
Figure 4: Transition selection and CE optimization.
Figure 5: Qualification of optimized transitions in the biological matrix using heavy synthetic peptides spiked in the serum sample (Steps 20–24).
Figure 6: Dilution curve.

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Acknowledgements

This work is supported by FNR (Luxembourg Fonds National de la Recherche) via a PEARL grant and by the Ministry of Higher Education and Research of Luxembourg via the PPM program. We thank S.-Y. Kim for bioinformatics help.

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Authors and Affiliations

  1. Luxembourg Clinical Proteomics Center, Centre de Recherche Public de la Santé, Strassen, Luxembourg

    Yeoun Jin Kim, Zaya Zaidi-Ainouch, Sebastien Gallien & Bruno Domon

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  1. Yeoun Jin Kim
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  2. Zaya Zaidi-Ainouch
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  3. Sebastien Gallien
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  4. Bruno Domon
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Contributions

Y.J.K. designed the experiments, analyzed data and wrote the manuscript. Z.Z.-A. performed the N-glycopeptide enrichment experiment. S.G. performed the SRM assay development, quantitative data analyses and prepared the manuscript. B.D. directed and supervised the project.

Corresponding author

Correspondence to Bruno Domon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Number of N-X-S/T tryptic peptides. (PPT 86 kb)

Supplementary Table 2

Set of reference peptides for retention time correction. (PPT 118 kb)

Supplementary Figure 1

MS/MS spectra of isotopically labeled synthetic peptide, AYLLPAPPAPGnASESEEDR* (n = asparagine with deamidation, R* = arginine with 13C/15N label. (a) Peptide fragmentation using collision induced dissociation (CID) in ion trap. (b) Peptide fragmentation using higher energy collision dissociation (HCD). (PPT 94 kb)

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Kim, Y., Zaidi-Ainouch, Z., Gallien, S. et al. Mass spectrometry–based detection and quantification of plasma glycoproteins using selective reaction monitoring. Nat Protoc 7, 859–871 (2012). https://doi.org/10.1038/nprot.2012.023

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