Dear Skyline Users:
We are pleased to announce the first Skyline User Group Meeting, which will be held in Vancouver, CA on Sunday afternoon before ASMS. We would like to thank the event sponsors (see below) for their generosity and interest in collaborating with the Skyline project on exciting new targeted and quantitative proteomics techniques. Thanks to them, the meeting attendance is free, though registration is required. We are also especially grateful to the investigators who have been deeply involved in helping to create some of the most exciting Skyline features now available in Skyline version 1.2, and who have agreed to speak about the research they are achieving with Skyline, described below.
--Brendan
When: May 20th, 2012
12:00 - 1:00 pm : Lunch served
1:00 - 3:30 pm : Presentations
3:30 - 4:30 pm : Snacks and break-out discussions
Where: Morris J Wosk Conference Center (http://tinyurl.com/Skyline-umg-2012-wosk), West Hastings Street, Vancouver, CA
Michael J. MacCoss, Ph. D. (University of Washington): Introduction and event host
Susan E. Abbatiello, Ph. D. (The Broad Institute): Effectively Dealing with Transition Selection and Data Analysis for Multiplexed Quantitative SRM-MS Assays across Multiple Vendor Instruments
Skyline has played a pivotal role in the success of the NCI’s Clinical Proteomics Technologies Assessment for Cancer consortium and our efforts in evaluating highly multiplexed quantitative assays for the verification of biomarker candidates in plasma. This presentation will focus on the use of Skyline in the context of CPTAC Verification studies as well as its routine and specialized use in the Proteomics Platform at the Broad Institute. (More info...)
Jarrett D. Egertson (MacCoss Lab, University of Washington): Multiplexed Data Independent Acquisition for Comparative Proteomics
A multiplexed data independent acquisition strategy is demonstrated on a Q-Exactive (Thermo Scientific). Five separate 4 m/z isolation windows are analyzed per MS/MS spectrum. These spectra are demultiplexed, during Skyline data import, into the five separate 4 m/z isolation windows, using a novel strategy with similarities to Hadamard multiplexing. The resulting data is then interpreted by Skyline in a manner similar to non-multipexed DIA experiments with a sampling frequency of 20x20 m/z wide windows but the specificity of an approach using 100x4 m/z wide windows. (More info...)
Christina Ludwig, Ph.D. (Institute of Molecular Systems Biology, ETH Zürich): Pinpointing phosphorylation sites using Selected Reaction Monitoring and Skyline
The precise and confident assignment of protein phosphorylation sites within a given peptide sequence still remains one of the major challenges in the field of phosphoproteomics. Typically, this issue becomes especially apparent when several possibly phosphorylated amino acids are located in close proximity. To test the utility of Selected Reaction Monitoring (SRM) as a method for specific phosphosite assignments we extensively investigated two metabolic proteins from Saccharomyces cerevisiae, which both show multiple possibly phosphorylated serine residues in very close proximity. All data analysis was performed manually using the software Skyline and exploiting its inherent capability to generate and optimize SRM assays for any type of phosphorylated (or generally modified) peptide and by predicting highly accurate, calibrated retention time information based on the iRT approach (independent retention time, www.Biognosys.ch). Finally we applied our assays onto phospho-enriched yeast samples for the unambiguous assignment and accurate quantification of endogenously occurring phosphopeptides. (More info...)
Brendan MacLean (MacCoss Lab, University of Washington): Status of the Skyline open-source software project four years after its inception
The Skyline project started just after ASMS 2008 as a 2-year effort to bring better SRM/MRM software tools to the NCI-CPTAC Verification Working Group that could support the variety of mass spectrometers in use in participating laboratories. Nearly 4 years later, the Skyline project is a thriving proteomics community open-source collaboration with solid support for another 4 years, hundreds of users and thousands of instances started each week. In this presentation, the Skyline lead software engineer will present recent developments and a roadmap for the project's future. (More info...)
Birgit Schilling, Ph.D. (Buck Institute for Research on Aging): Platform Independent and Label-free Quantitation of Protein Acetylation and phosphorylation using MS1 Extracted Ion Chromatograms in Skyline
We expanded the capabilities of Skyline to process full-scan mass spectral data (MS1) proteomic experiments so that ion intensity chromatograms of many peptide analytes could be integrated across HPLC MS/MS experiments. The Skyline MS1 Filtering label-free quantitative approach provides key graphical tools to support interrogation of multiple acquisitions for MS1-based peptide abundance, including visual inspection of peak picking, visualization of underlying MS/MS spectra that were sampled, and both automated and manual integration. (More info...)
Andrew B. Stergachis (Stamatoyannopoulos Lab, University of Washington): Rapid empirical identification of optimal peptides for targeted proteomics
We report a method for high-throughput, cost-efficient empirical discovery of optimal proteotypic peptides and fragment ions for targeted proteomics applications using in vitro–synthesized proteins and the Skyline software package. We demonstrate the approach using human transcription factors, which are typically difficult, low-abundance targets and empirically derived proteotypic peptides for 98% of the target proteins. We show that targeted proteomic assays developed using our approach facilitates robust in vivo quantification of human transcription factors. (More info...)
J. Will Thompson, Ph. D. (Duke University Proteomics Core Facility): Using Skyline to Monitor Long-Term Performance Metrics of High-Resolution Mass Spectrometers
Skyline and full-scan MS2 filtering has been utilized over a 1 year period to monitor key system suitability metrics of three QToF mass spectrometers in use in a high-throughput proteomics core environment. Key metrics tracked over this period include retention time reproducibility, chromatographic peak shape, peak intensity, and MS2 fragmentation. The importance and ease of implementing a system suitability practice for high-resolution instruments using Skyline will be discussed. (More info...)
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