title: Constrained selected reaction monitoring: quantification of selected post-translational modifications and protein isoforms.
abstract: Selected reaction monitoring (SRM) is a mass spectrometry method that can target signature peptides to provide for the detection and quantitation of specific proteins in complex biological samples. When quantifying a protein, multiple peptides are generated using a specific protease such as trypsin, thereby allowing a choice of signature peptides with robust signals. In contrast, signature peptide selection can be constrained when the goal is to monitor a specific post-translational modification (PTM) or protein isoform, as the signature peptide must include the amino acid residue(s) of PTM attachment or sequence variation. This can force the selection of a signature peptide with a weak SRM response or one that is confounded by high background. In this article, we discuss steps that can be optimized to maximize peptide selection and assay performance of constrained SRM assays, including tuning instrument parameters, fragmenting product ions, using a different protease, and enriching the sample. Examples are provided for phosphorylated or citrullinated peptides and protein isoforms.
authors.affiliation: Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Liu
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Jin
authors.forename: Richard
authors.initials: R
authors.lastname: O'Brien
authors.forename: Joan
authors.initials: J
authors.lastname: Bathon
authors.forename: Harry C
authors.initials: HC
authors.lastname: Dietz
authors.forename: Eric
authors.initials: E
authors.lastname: Grote
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes; D002417:Cattle; D002956:Citrulline / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis*; D008969:Molecular Sequence Data; D051579:Neurogranin / Q000032:analysis*; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000032:analysis*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods / Q000592:standards*; D059629:Signal-To-Noise Ratio; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*; D014357:Trypsin / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D020033:Protein Isoforms; D051579:Neurogranin; D002956:Citrulline; D048052:Mitogen-Activated Protein Kinase 3; D014357:Trypsin