Philip Remes, Ph.D., is a senior research scientist in the R&D department of Thermo Fisher Scientific in San Jose, California. He received his Ph.D. in Gary Glish’s lab at the University of Chapel Hill, North Carolina, where he worked on quadrupole ion trap and mass filter instrumentation. At Thermo, he worked with Jae Schwartz on instrument control software, ion trap analyzers, detectors and simulations for the LTQ Velos platform. Later he did similar work as a part of Mike Senko’s Tribrid development team on the Fusion, Lumos, and Eclipse projects. In recent years, Philip has continued working on ion trap technology, branching out to learn about applications and get first-hand experience in how they can benefit from improvements in hardware, instrument control, signal processing, and informatics.
Targeted MS/MS is the gold standard for quantitative quality in proteomics but is not widely utilized for large assays (1000’s of targets), because of the manual work involved in setting up the experiment, maintaining the retention time schedule, and analyzing the results. We created a Skyline External tool that automates the tedious parts and makes large scale targeted MS/MS a competitive option to Data Independent Acquisition (DIA) for some situations.
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The tool starts from imported peptide DIA search results, refines the transitions, and creates an instrument targeted MS/MS method file that accounts for the details of the LC peak width and the instrument acquisition speed, be it for Orbitrap, Ion trap, or Triple quad analysis. We also show how the tool interfaces with our new real-time chromatogram alignment technology, which eliminates the problem of maintaining the assay schedule over time and provides a significant boost to the number of feasible targets by narrowing the scheduling windows. The automated peak-picking in Skyline is aided by the transition refinement, the peptide search library, and the narrow scheduling windows, making it extremely reliable. With Skyline, our tool, and real-time chromatogram alignment, we demonstrate the creation and use of a targeted MS/MS assay for > 2000 targets in a 30 minute gradient.
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