title: Biomarker verification using selected reaction monitoring and shotgun proteomics.
abstract: Shotgun proteomics (liquid chromatography-electrospray ionization-mass spectrometry, LC-ESI-MS/MS) has dominated the strategies for global protein expression in subcells, cells, tissues, and whole organisms with several types of approaches, as isobaric tags for relative and absolute quantification (iTRAQ), isotope-coded affinity tags (ICAT), or stable isotope labeling using amino acids in cell culture (SILAC) and non-labeling (label free) methods. Shotgun proteomics practically replaced the classical 2D gel electrophoresis. Selected reaction monitoring (SRM), also denominated multiple reaction monitoring (MRM), is a targeted quantitative technology that uses a complex mixture of tryptic peptides that can be selectively detected by liquid chromatography coupled to electrospray triple-quadrupole mass spectrometer; this system can select precursor ions in combination with their correspondent product ions during collision-induced dissociation to produce specific detection related to a particular protein. Here we describe protocols that are efficient to produce a complete enzymatic trypsin digestion from complex biological matrices and concomitant material to be used for LC-SRM-MS and LC-ESI-MS/MS (labeled or label free).
authors.affiliation: Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.
authors.forename: Angel Mauricio
authors.initials: AM
authors.lastname: Castro-Gamero
authors.forename: Clarice
authors.initials: C
authors.lastname: Izumi
authors.forename: José César
authors.initials: JC
authors.lastname: Rosa
mesh_terms: D000345:Affinity Labels; D015415:Biomarkers; D002853:Chromatography, Liquid; D007553:Isotope Labeling; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000345:Affinity Labels; D015415:Biomarkers