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), isotopecoded affinity tags (ICAT), or stable isotope labeling using amino acids in cell culture (SILAC) and nonlabeling (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 collisioninduced 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).