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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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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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.
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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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DOI: https://doi.org/10.1038/nprot.2012.023
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