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      Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results*


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          Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.

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          Most cited references 57

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          A global protein kinase and phosphatase interaction network in yeast.

          The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses.
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            OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data.

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              Tracking cancer drugs in living cells by thermal profiling of the proteome.

              The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity. Copyright © 2014, American Association for the Advancement of Science.

                Author and article information

                Mol Cell Proteomics
                Mol. Cell Proteomics
                Molecular & Cellular Proteomics : MCP
                The American Society for Biochemistry and Molecular Biology
                December 2017
                25 October 2017
                25 October 2017
                : 16
                : 12
                : 2296-2309
                [1]From the ‡Biognosys, Wagistrasse 21, 8952 Schlieren, Switzerland.
                [2]§Thermo Fisher Scientific, 28199 Bremen, Germany.
                [3]¶Somatosensory Signaling and Systems Biology Group, Max Planck Institute of Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Goettingen, Germany
                Author notes
                ‖ To whom correspondence should be addressed: Biognosys, Wagistrasse 21, 8952 Schlieren, Switzerland, Tel.: +41-(0)44-738-20-40, Fax: +41-(0)44-738-20-49, E-mail: lukas.reiter@ 123456biognosys.com .
                © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

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