Table of Contents

guest
2020-10-29
Speakers
   Joseph Brown, Ph.D.
   Christine Carapito, Ph.D.
   Josh Eckels
   Jarrett Egertson, Ph.D.
   Andy Hoofnagle, MD, Ph.D.
   Jacob D. Jaffe, Ph.D.
   Brendan MacLean
   Brett Phinney, Ph.D.
   Matthew J. Rardin, Ph.D.
   Olga Schubert

Speakers


Ten speakers with interesting and different areas of expertise in Skyline use and development agreed to speak at the Skyline User Group Meeting at ASMS 2013 in Minneapolis, MN. Their presentations were recorded, and have now been posted in various formats for your review.

 Josheph N. Brown, Ph.D.
 Christine Carapito, Ph.D.
 Josh Eckels
 Jarrett D. Egertson, Ph.D.
 Andy Hoofnagle, MD, Ph.D.
 Jacob D. Jaffe, Ph.D.
 Brendan MacLean
 Brett S. Phinney, Ph.D.
 Matthew J. Rardin, Ph.D.
 Olga Schubert



Joseph Brown, Ph.D.


Joseph Brown Joseph Brown, Ph.D., is a senior scientist in the Biological Sciences Division at the Pacific Northwest National Laboratories. His work focuses on using high-throughput “-omic” technologies to investigate the global response to pathogenic viral infections within humans and nonhuman primates. Joseph earned a BS in molecular biology at the University of West Florida. In 2002, he joined the laboratory of Dr. Maureen Goodenow at the University of Florida, studying the response of primary monocyte-derived macrophages to HIV-1 treatment/infection. He then extended his research to the proteomics realm when he joined Dick Smith’s group at PNNL as a post-doctoral fellow studying a number of viruses, including monkeypox, influenza, and HIV-1.

Effective design and analysis of multiplexed quantitative SRM data with Skyline

The first phase of a targeted quantitative study includes selection and optimization. These early decisions are the most crucial as they influence the entirety of the downstream process. This presentation will focus on how we have used the Skyline software package to effectively guide these initial phases for two large-scale NCI projects: CPTAC and EDRN.

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Christine Carapito, Ph.D.


Christine Carapito Christine Carapito, Ph.D., works on developing mass spectrometry-based quantitation methods and applying them to biological systems, especially using targeted workflows. After an engineer degree in Biotechnology, she earned her PhD in Analytical Chemistry at the University of Strasbourg in the lab of Dr. Alain Van Dorsselaer in 2006, specializing in mass spectrometry-based method development for the characterization of biomolecules. She then joined Ruedi Aebersold’s group at ETH Zürich, where she worked as post-doctoral fellow on glycopeptides enrichment methods coupled to ETD fragmentation and on the generation of the human SRM atlas. In 2010, she obtained a CNRS permanent Research Scientist position at the Bioorganic Mass Spectrometry Laboratory in Strasbourg (Director: Dr. Van Dorsselaer).

Developing, transferring, sharing, combining, and bridging global and targeted quantitative methods and data in a platform-independent manner thanks to Skyline

When working on biological applications for which MS data have been acquired on various LC-MS/MS and LC-SRM platforms (ABSciex, Agilent Technologies, Bruker Daltonics, Thermo Scientific and Waters in our case), software tools available to combine and visualize those data in a platform-independent manner play a crucial role. We are using Skyline to develop targeted SRM assays (choose proteotypic peptides from libraries, optimize transitions, …), extract quantitative results from data dependent LC-MS/MS acquisitions and bridge those global and targeted quantitative results. We are also extensively using the retention time calibration/prediction functionalities to transfer methods from one to another platform and optimize chromatographic conditions.

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Josh Eckels


Josh Eckels Josh Eckels has lead development on all proteomics related projects for LabKey Server over the past 7 years, and he has made invaluable contributions to the Panorama project over the past year. He is experienced in security-related projects, client-side end user applications, and server-side development. Prior to joining LabKey, Josh worked most recently on Amazon.com's data warehouse, developing the software that schedules and executes loading and querying of more than 30 terabytes of data. Before that, he was the team lead for the debugger for BEA System's WebLogic Workshop. At LabKey, he focuses on mass spectrometry, experimental annotations and general infrastructure. Josh has a BS in Computer Engineering from Northwestern University.

Panorama: targeted proteomics repository software for Skyline

Panorama is an open-source and freely available repository server application for targeted assays that is designed to facilitate sharing and querying across large pools of targeted proteomics experiments. It has been implemented as a module in LabKey Server, a biomedical data management platform with rich support for proteomics data. LabKey Server provides a flexible and secure infrastructure for data sharing, ease of deployment, and the ability to incorporate other biological information supported in the LabKey environment. Panorama is tightly integrated into a Skyline-based proteomics workflow allowing researchers to publish documents to a Panorama server, mark assays as representative, and download libraries containing data from validated assays that can be used for designing new targeted methods, or comparing with newly acquired data.

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Jarrett Egertson, Ph.D.


Jarrett D. Egertson Jarrett Egertson, Ph.D., is a postdoctoral researcher at the University of Washington Department of Genome Sciences. He works in the MacCoss Lab and primarily focuses on developing new data acquisition methods and software in support of these methods. Jarrett earned his undergraduate degree (B.S. in Molecular, Cell, and Developmental Biology) from UCLA in 2008. While earning his undergraduate degree, Jarrett researched at the Spielberg Family Center for Applied Proteomics at the Cedars-Sinai Medical Center.

Application of data independent acquisition techniques optimized for improved precursor specificity

Novel data independent acquisition (DIA) techniques with improved precursor specificity are presented. The optimization of these techniques for samples of varying complexity will be discussed along with applications to the study of aging in yeast. Another study will be presented comparing the performance of multiple novel and pre-existing DIA techniques on a Q-Exactive. The presentation will end with a brief tutorial on using Skyline to implement these techniques and analyze the resulting data.

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Andy Hoofnagle, MD, Ph.D.


Andy Hoofnagle Andy Hoofnagle, MD, Ph.D., is an Associate Professor in the Department of Laboratory Medicine at the University of Washington. His laboratory focuses on developing CLIA-certified assays for small molecules using mass spectrometry. They also investigate the function of high density lipoproteins in patients at increased risk for cardiovascular disease and the translation of proteomics technologies into the clinical laboratory.

Using Skyline for Lipidomics

Our interest in lipidomics is grounded in the likelihood that lipids are important mediators of disease via their influence on macrophages and endothelial cells. We have a special focus on the sphingolipids due to their fundamental role in cell signaling. Our laboratory has developed a novel single-step extraction method to quantify more than 100 lipids from high density lipoproteins. Current software solutions for peak integration and data handling are limited. We have identified a workaround in Skyline that allows for the rapid analysis of many samples simultaneously. The solution takes advantage of the excellent peak identification and integration capabilities in Skyline and has greatly simplified the workflow for lipidomics in our laboratory.


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As of Skyline 3.1, native small molecule targeting has been added. (See Small Molecule Targets tutorial)

 

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Jacob D. Jaffe, Ph.D.


Jacob D. Jaffe Jacob D. Jaffe, Ph.D., is the Assistant Director of the Proteomics Platform at the Broad Institute. He obtained his B.A. in Biochemistry from the University of Pennsylvania and his Ph.D. from Harvard University where he studied with George Church and Howard Berg. Dr. Jaffe has pioneered diverse problems in modern proteomics including large-scale mapping of proteomic data onto genomes, thus allowing their de novo annotation from proteomic evidence, pattern recognition for quantitative proteomics, determination and quantification of epigenetic marks on histone proteins, and high-throughput targeted phosphoproteomics.

Discovery to Targets for a Phosphoproteomic Signature Assay: One-stop shopping in Skyline

The effective dimensionality of the phosphoproteome is actually much smaller than the number of phosphosites due to coordinate regulation by a limiting number of kinases and phosphatases. To study these properties, we generated a large perturbational phosphoproteomics data set across multiple cell types and compounds using SILAC methodologies. Initial analysis of the data suggests that there are indeed groups of coordinately regulated sites that could be condensed into clusters. Some response clusters were cell-line independent, and some clusters showed responses that clearly represent similar activities by structural analogs. This source dataset may be the largest perturbational phosphoproteomic dataset in existence.

We seek to collapse the phosphoproteome into a limited number of sites that can be monitored in a targeted MS-based assay. This assay will be used to generate data for the Library of Integrated Cellular Signatures (LINCS) consortium. We call this assay the P100 assay, as an homage to the L1000 assay which achieves dimensionality reduction for gene expression. We collapsed the data into 55 clusters, and picked two exemplary phosphopeptides from each for the configuration of the assay. We have synthesized these peptides, their non-phosphorylated cognates, and distal peptides from the same protein so that we may determine stoichiometry.

Skyline is our tool of choice for rapidly coöpting discovery data initially processed with MaxQuant into a framework for a targeted this assay, and we will distribute assay parameters and aggregate cross-laboratory results via Panorama.


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Brendan MacLean


Brendan MacLean Brendan MacLean worked at Microsoft for 8 years in the 1990s where he was a lead developer and development manager for the Visual C++/Developer Studio Project.  Since leaving Microsoft, Brendan has been the Vice President of Engineering for Westside Corporation, Director of Engineering for BEA Systems, Inc., Sr. Software Engineer at the Fred Hutchinson Cancer Research Center, and a founding partner of LabKey Software.  In this last position he was one of the key programmers responsible for the Computational Proteomics Analysis System (CPAS), made significant contributions to the development of X!Tandem and the Trans Proteomic Pipeline, and created the LabKey Enterprise Pipeline.  For the last 4 and a half years he has worked as a Sr. Software Engineer within the MacCoss lab and been responsible for all aspects of design, development and support in creating the Skyline Targeted Proteomics Environment and its growing worldwide user community.

Status of the Skyline open-source software project 5 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 5 years later, the Skyline project is a thriving proteomics community open-source collaboration with solid support for another 3 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. Topics covered will include:
  • Advances in chromatogram extraction from full-scan acquisition data
  • Expanding support for new instrument vendors and new instruments
  • New peak picking algorithm based on mProphet, with probability based confidence metrics
  • Improved support for interaction with external tools like QuaSAR and MSstats
  • Experimental meta data and statistics in Skyline

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Brett Phinney, Ph.D.


Brett Phinney Brett Phinney, Ph.D., has been the manager of the Proteomics Core at UC Davis Genome Center, University of California since 2005 where he both uses Skyline in his research and has taught courses to help others learn more about using Skyline for targeted proteomics. His prior work experience includes: Research Assistant Professor, Michigan Proteome Consortium, Michigan State University, 2004 - 2005. Director, Michigan State University Proteomics Facility, Michigan State University, 2002 - 2005. Research Associate, Michigan Proteome Consortium, Michigan State University, 2001 - 2002. Postdoctoral Fellow, Michigan State University, 2000 - 2001.

Using Skyline to analyze the SPRG2013-2014 Targeted Proteomics Standard

Proteomics technologies are an integral part of biological and clinical research. Significant contributions by the proteomics field are driven by the diverse and advanced analytical approaches employed to comprehensively characterize proteomes, including quantitative analysis of proteome variations, modifications, and interactions. The ABRF Proteomics Standards Research Group (sPRG) functions to design and develop performance standards and resources for mass spectrometry-based proteomics applications. The sPRG is currently conducting a study focused on generation of a standard for interassay, interspecies, and interlaboratory normalization in label-free as well as in quantitative stable-isotope label-based analyses. The standard has been formulated as two mixtures: 1000 stable isotope 13C/15N-labeled synthetic tryptic peptides alone, and peptides mixed with a tryptic digest from HEK 293 cell lysate. This presentation will detail how the sPRG used skyline to analyze this standard and will present several of the challenges we encountered when analyzing isotope labeled peptide standards in complex matrices.

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Matthew J. Rardin, Ph.D.


Matthew J. Rardin Matthew J. Rardin, Ph.D., is currently a postdoctoral fellow in the laboratory of Dr. Brad Gibson at the Buck Institute for Research on Aging. Originally from Colorado, he received a B.S. in Microbiology at Colorado State University and obtained a Ph.D. in 2008 from the University of California San Diego in the laboratory of Dr. Jack Dixon where he studied the role of phosphorylation in mitochondria. His postdoctoral work in the Gibson lab is focused on developing techniques for the identification and quantitation of proteins and post-translational modifications in a variety of mitochondrial paradigms using mice as a model system. Recently his work has focused on the use of label free quantitation including MS1-Filtering and SWATH for studying the role of lysine acetylation in mitochondria from SIRT3 knockout animals and how it regulates signaling mechanisms within this dynamic organelle.

Label free quantitation of proteomic data using MS1 Filtering and MS/MSALL with SWATH acquisition

Recently, we developed a label free quantitation tool called MS1 Filtering using expanded features in Skyline. MS1 Filtering processes precursor ion intensity chromatograms of peptide analytes from full scan mass spectral data acquired during data dependent acquisitions by HPLC MS/MS. In contrast data independent acquisitions such as SWATH can quantify product ion analytes from the MS2 scan when a spectral library is available. Interestingly, during each SWATH acquisition cycle on the Triple TOF 5600 mass spectrometer an MS1 scan is acquired providing two independent methods for quantifying analytes in a single acquisition or experiment. We have carried out a series of experiments to examine the utility of using both methods for interrogation of proteomic data sets. This presentation will focus on our efforts to explore the utility of MS1 and MS2 quantitation, either alone or in combination, using Skyline.

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Olga Schubert


Olga Schubert Olga Schubert studied Biology at ETH Zurich (Switzerland). During her Masters she focused on cell biology and cancer research. Afterwards, she spent six month in Italy as a trainee at EMBL Monterotondo to gain more insights into mouse biology and neuroscience. In April 2010 she joined the group of Prof. Ruedi Aebersold at the Institute of Molecular Systems Biology, ETH Zurich, where she has been applying mass spectrometry to study the proteome of Mycobacterium tuberculosis. Recently, she developed the Mtb Proteome Library, a database containing quantitative assays for targeted mass spectrometry for all proteins of Mycobacterium tuberculosis. Her current research focus is on proteome-wide absolute quantification of Mycobacterium tuberculosis using SRM and data-independent acquisition (SWATH-MS).

Development and application of assays for targeted mass spectrometric analysis of the complete proteome of Mycobacterium tuberculosis

Worldwide, two billion people are infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To facilitate basic and translational research into Mtb we developed a library of quantitative assays for the targeted mass spectrometric analysis of the Mtb proteome by selected reaction monitoring (SRM). The software tools Skyline and mProphet were used extensively in creating a resource that contains high quality SRM assays for 97% of the 4,012 proteins annotated in Mtb, of which 72% could be validated in whole cell lysates. From this proteome-wide SRM data, the absolute abundances of 55% of all Mtb proteins were estimated, revealing a dynamic range of the Mtb proteome of over four orders of magnitude. We used the thus generated assay library to gain protein-level insights into the dynamic regulation of almost the entire dormancy survival regulon in response to hypoxia by SRM. Currently, we are extending and optimising the library for targeted data extraction of MS/MS spectra generated by data-independent acquisition (SWATH-MS), which allows us to quantify hundreds of proteins in a single run. In conclusion, we present the development and application of a library of publicly available, quantitative assays for targeted proteomics by SRM and SWATH-MS to accurately monitor abundance changes of virtually all Mtb proteins with high sensitivity and reproducibility.

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