PMID- 26700042 OWN - NLM STAT- MEDLINE VI - 1394 TI - A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses. PG - 75-85 LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PL - United States TA - Methods Mol Biol JT - Methods in molecular biology (Clifton, N.J.) JID - 9214969 IS - 1940-6029 (Electronic) LID - 10.1007/978-1-4939-3341-9_6 [doi] FAU - Moradian, Annie AU - Moradian A AD - Proteome Exploration Laboratory, California Institute of Technology, BI 211, MC139-74, Pasadena, CA, 91125, USA. FAU - Porras-Yakushi, Tanya R AU - Porras-Yakushi TR AD - Proteome Exploration Laboratory, California Institute of Technology, BI 211, MC139-74, Pasadena, CA, 91125, USA. FAU - Sweredoski, Michael J AU - Sweredoski MJ AD - Proteome Exploration Laboratory, California Institute of Technology, BI 211, MC139-74, Pasadena, CA, 91125, USA. FAU - Hess, Sonja AU - Hess S AD - Proteome Exploration Laboratory, California Institute of Technology, BI 211, MC139-74, Pasadena, CA, 91125, USA. shess@caltech.edu. IS - 1064-3745 (Linking) RN - 0 (Peptides) RN - 0 (Proteins) RN - 0 (Proteome) SB - IM MH - Chromatography, Liquid MH - HeLa Cells MH - Humans MH - Peptides MH - Proteins MH - *Proteome MH - Proteomics/*methods MH - Tandem Mass Spectrometry OTO - NOTNLM OT - MRM OT - Quadrupole mass spectrometry OT - Quantitation DCOM- 20161013 LR - 20170519 DP - 2016 AB - When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken into account, setting up an MRM assay is relatively straightforward. Typically, proteotypic peptides with favorable mass spectrometric properties are synthesized with a heavy isotope for each protein that is to be monitored. Retention times and calibration curves are determined using triple-quadrupole mass spectrometers. The use of iRT peptide standards is both recommended and fully integrated into the bioinformatics pipeline. Digested biological samples are mixed with the heavy and iRT standards and quantified. Here we present a generic protocol for the development of an MRM assay.