title: Evaluation of absolute peptide quantitation strategies using selected reaction monitoring.
abstract: The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.
authors.affiliation: Proteome Sciences, Cobham, Surrey, UK. james.campbell@proteomics.com
authors.forename: James
authors.initials: J
authors.lastname: Campbell
authors.forename: Taha
authors.initials: T
authors.lastname: Rezai
authors.forename: Amol
authors.initials: A
authors.lastname: Prakash
authors.forename: Bryan
authors.initials: B
authors.lastname: Krastins
authors.forename: Loïc
authors.initials: L
authors.lastname: Dayon
authors.forename: Malcolm
authors.initials: M
authors.lastname: Ward
authors.forename: Sarah
authors.initials: S
authors.lastname: Robinson
authors.forename: Mary
authors.initials: M
authors.lastname: Lopez
mesh_terms: D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000097:blood / Q000737:chemistry; D006801:Humans; D007554:Isotopes; D057230:Limit of Detection; D010455:Peptides / Q000032:analysis* / Q000592:standards; D040901:Proteomics / Q000592:standards* / Q000706:statistics & numerical data; D011786:Quality Control; D012015:Reference Standards
chemical_list: D001053:Apolipoproteins; D007554:Isotopes; D010455:Peptides