Journal
NATURE PROTOCOLS
Volume 7, Issue 5, Pages 859-871Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2012.023
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Funding
- FNR (Luxembourg Fonds National de la Recherche) via a PEARL
- Ministry of Higher Education and Research of Luxembourg via the PPM
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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 (SRSRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRSRM 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 SRSRM 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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