title: Biomarker validation in blood specimens by selected reaction monitoring mass spectrometry of N-glycosites.
abstract: Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as the method of choice for the validation in blood serum, plasma, or other clinically relevant specimens of biomarker candidates arising from comparative proteomics or other discovery strategies. Here, we describe a method in which N-glycosites are selectively enriched from biological specimens by solid phase capture and PNGase F release, and then analyzed by SRM. Focusing the highly sensitive targeted mass spectrometry method on a subproteome enriched for secreted and shed proteins reproducibly identifies and quantifies such proteins in serum and plasma at the low nanogram per milliliter (ng/mL) concentration range. This protocol is intended to give an introduction to SRM-based targeted mass spectrometry with a special focus on the validation of biomarker candidates.
authors.affiliation: Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Reto
authors.initials: R
authors.lastname: Ossola
authors.forename: Ralph
authors.initials: R
authors.lastname: Schiess
authors.forename: Paola
authors.initials: P
authors.lastname: Picotti
authors.forename: Oliver
authors.initials: O
authors.lastname: Rinner
authors.forename: Lukas
authors.initials: L
authors.lastname: Reiter
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D015415:Biomarkers / Q000097:blood* / Q000737:chemistry; D001800:Blood Specimen Collection / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D019151:Peptide Library; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers; D019151:Peptide Library