title: Immuno-mass spectrometry: quantification of low-abundance proteins in biological fluids.
abstract: Mass spectrometry is emerging as one of the most promising analytical techniques to examine simultaneously hundreds of analytes quickly, precisely, and accurately, using minute sample volumes. Currently, a major bottleneck in the verification phase of putative biomarkers is the lack of methods/reagents to quantify low levels of analytes in biological fluids. A major objective is to establish a high-throughput multiple reaction monitoring (MRM) assay capable of quantifying low-abundance proteins or peptides in biological fluids (low μg/L range) using mass spectrometry. The experimental procedure we propose, called immuno-mass spectrometry, consists of immuno-capturing analytes of interest from relevant biological fluids in 96-well microtiter plates and performing in-well tryptic digestion, with subsequent MRM of digested peptides on a triple quadrupole mass spectrometer. With such a strategy, limits of detection of 0.1-1 μg/L proteins in serum with a coefficient of variation of <20% can be obtained. This methodology could be adapted quickly and easily to potential candidates of interest, thus providing a much needed technology to bridge the gap between discovery and validation platforms.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Vathany
authors.initials: V
authors.lastname: Kulasingam
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Smith
authors.forename: Ihor
authors.initials: I
authors.lastname: Batruch
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D001826:Body Fluids / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis* / Q000276:immunology*; D015203:Reproducibility of Results; D013223:Statistics as Topic
chemical_list: D010455:Peptides; D011506:Proteins