Table of Contents

guest
2020-07-09
Speakers
   Susan Abbatiello
   Michael MacCoss
   Matthew MacDonald
   Brendan MacLean
   Sarah Michaud
   Bhavin Patel
   Pawel Sadowski
   Birgit Schilling
   Tobias Schmidt
   Selene Swanson
   Sebastian Vaca

Speakers


Ten speakers with interesting and different areas of expertise in Skyline use and development spoke at the Skyline User Group Meeting at ASMS 2019 in Atlanta, GA.

* PDFs of the presentations are posted on each speaker's page below:

Susan Abbatiello, Ph.D.

Matthew MacDonald, Ph.D.

Brendan MacLean

Sarah Michaud, Ph.D.

Bhavin Patel, MD

Powel Sandowski, Ph.D.

Birgit Schilling, Ph.D.

Tobias Schmidt

Selene Swanson

Sebastian Vaca, Ph.D.




Susan Abbatiello


Susan Abbatiello Susan Abbatiello Ph.D., received a Ph.D. in analytical chemistry at the University of Florida, and completed a postdoc at the University of Pittsburgh’s Hillman Cancer Center. Susan is currently the executive director of the MS Core Facility at the Barnett Institute at Northeastern University, and a visiting scientist in the Proteomics Platform at the Broad Institute of MIT and Harvard. Susan’s research focuses on the development of experiments to measure potential protein biomarkers in cell lines and blood for diseases such as cancer. Susan has co-chaired a committee that is part of the National Cancer Institute’s Clinical Proteomic Technology Assessment for Cancer (CPTAC).

Skyline: 10-year Retrospective

Ten years ago a group of about 20 dedicated triple quadrupole operators met at the Broad Institute to learn about the features of Skyline -- this modest beginning evolved into the Skyline User Group Meeting -- the reason you are here today. Nowadays, the Skyline user base has grown significantly with 94,000 new installations of Skyline since 2009. Birgit and I will provide an entertaining retrospective over the last 10 years – what were its key achievements? Read More
How did Skyline bring results from labs together even when they used different instrumentation? By keeping its format, vendor-neutral, Skyline has developed great support from all the instrument vendors. In the initial days, we used Skyline for SRM supporting CPTAC projects, system suitability and biomarker research. Over time, the development team added new scan-type capabilities, such as MS1-Filtering, PRM and DIA and now even support for small molecule workflows! Great new features were implemented including iRT (indexed retention time), tool store connections and even linking Skyline with Panorama – the online repository for Skyline research data! A truly comprehensive quantitative environment for everybody, everywhere in the world!


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Michael MacCoss


Mike MacCoss Michael MacCoss Mike became interested in biomedical applications of mass spectrometry while working in Dr. Patrick Griffin’s protein mass spectrometry lab at Merck Research Laboratories. He obtained a Ph.D. with Professor Dwight Matthews and pursued a postdoc with Professor John R. Yates III. In 2004 he started the MacCoss lab at the University of Washington and it became obvious that while mass spectrometry data could be collected quickly and robustly, the lack of computational tools for the visualization and analysis of these data was a stumbling block. In 2008 he hired Brendan MacLean with the goal of developing professional quality software tools for quantitative proteomics. Read More
Mike has worked closely with the Skyline development team and our outstanding group of laboratory scientists and collaborators to ensure that our software uses analytical approaches that have been thoroughly vetted by the mass spectrometry community.


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Matthew MacDonald


Matthew MacDonald Matthew MacDonald ,Ph.D. Throughout his graduate and postdoctoral training to his current position as an assistant professor of psychiatry, University of Pittsburgh, MacDonald's main research focus has been validating and utilizing proteomic approaches to investigate synaptic protein network dysfunction in postmortem brain tissue from patients with neuropsychiatric disease. As a postdoctoral fellow (with Drs. Robert Sweet and Nathan Yates) and now a faculty member he has continued to push the envelope of proteomics approaches in human postmortem brain tissue to increase proteomic depth, throughput and spatial resolution.

Multi-omics Approach Identifies Pathological Phosphorylation Events Driving Synapse Loss in Schizophrenia

Schizophrenia is a complex trait disorder in which genetic risk factors converge on synaptic protein networks, altering synaptic architecture, and impairing brain function. Synaptic protein network features include protein expression, trafficking, and activity. To assess interactions between these features, we utilized microscopy as well as targeted and label free mass spectrometry to quantify synapse number and size as well as protein expression, synaptic protein levels, and protein phosphorylation in cortical tissue from 50 schizophrenia and 50 matched control subjects. Read More
To enhance the value of these data sets generated with skyline, we utilized co-regulation network and cross-network analyses; identifying a set of altered phosphorylations on f-acting binding and glutamate receptor trafficking proteins strongly correlated with both synaptic protein level alterations and synapse loss in schizophrenia. CRISPR-Cas9 was used to generate phosphomimetic mice for select phospho-alterations and confirm their causality in proteome and spine pathology.


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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 9 and a half years he has worked as a Sr. Software Engineer within the MacCoss lab and lead 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 10 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 10 years later, the Skyline project is a thriving proteomics community open-source collaboration supporting 6 mass spec instrument vendors, integrated with a wide variety of external software, with thousands of users worldwide and many thousands of instances started each week. Read More
In this presentation, the Skyline principal developer will present recent developments and a roadmap for the project's future. Topics covered will include:
  • New! Audit logging
  • New! Small molecule user interface
  • Advances in calibrated quantification
  • Improved command-line support running on Linux
  • Growth in the Skyline software ecosystem for targeted MS (Skyline, Panorama, and External Tools)
  • Strong industry support from instrument vendors
  • Courses, workshops, webinars and other efforts to create instructional resources for the Skyline community


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Sarah Michaud


Sarah Michaud Sarah Michaud received her M.Sc. in Genome Sciences and Technology at the University of British Columbia, where she investigated quantitative proteomics of honeybees during viral infection. She began her work developing targeted assays for quantitation of mouse plasma proteins at MRM Proteomics in 2014, and is now a project manager at the UVic Genome BC Proteomics Center in Victoria, Canada, where her focus is the development of 3,000 MRM assays in 20+ mouse tissues.

Development of Quantitative MRM Assays for the Measurement of 3,000 Proteins across 20 Mouse Tissues

An ongoing effort at our Centre is the development of MRM-MS assays quantifying 3,000 proteins across 20 mouse tissues for use in molecular phenotyping. Our assays use stable isotope-labelled standard peptides for quantitation and undergo rigorous optimization of peptide- and instrument-specific parameters followed by assay development and validation according to CPTAC guidelines. Assay development determines the LLOQ and linear range of the assay via standard curve, and assay variability is calculated over 5 days at three concentrations spanning the final range of the assays. Read More
To date assays have been developed and validated for 1643 unique proteins in 11 mouse tissues. Once developed, assays are multiplexed into panels of 125 proteins and used to measure normal tissue protein concentrations in 3 common mouse strains. Data processing and analysis for all steps, from optimization to sample measurement, was performed using Skyline, and is a critical step in our pipeline for large scale assay development.


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Bhavin Patel


Bhavin Patel Bhavin Patel , MD. works as a senior research and development scientist in MS reagents group at Thermo Fisher Scientific. I have a medical Doctor degree from the MS University in India, and a masters in biotechnology from the Georgetown University. I had led a protein biomarker targeted MS assay group at NextGen Sciences and a proteomics/mass spectrometry group at Fox Chase Cancer Center before joining Thermo Fisher Scientific in 2013. Since joining Thermo, I have led the development of targeted MS assay kits, new MS system suitability standards, protein sample preparation reagents, antibody validation method for IP-MS, and reagents for targeted quantitative proteomic analysis.

Targeted Mass Spectrometry Assays for Absolute Quantitation of AKT/mTOR Signaling Pathway Proteins

Proteomics offers a deeper understanding of the protein events influencing cellular and biological function. However, Identifying key regulated proteins and their implicated signaling pathways represents a major analytical challenge to researchers. We have optimized a multiplex immunoprecipitation to targeted mass spectrometry (mIP-tMS) workflow to develop the SureQuant pathway panels, achieving simultaneous enrichment and absolute quantitation of multiple total and phosphorylated proteins from the AKT/mTOR pathway. Read More
We discuss improved enrichment/sample preparation method as well as development and validation of targeted MS assay including calibration curves, robustness, precision and accuracy of these assays. We have utilized the Skyline software for assay development and data analysis for absolute quantitation of AKT pathway proteins. Additionally, we have developed a system suitability standard containing isotopologue peptides mixture and utilized Skyline for data analysis. Finally, we’ll examine applications of multiplexed IP to targeted MS assays compared to existing immunoassay techniques.


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Pawel Sadowski


Pawel Sadowski Pawel Sadowski, Ph.D. completed his Ph.D. in the field of quantitative proteomics at the University of Cambridge. During subsequent post-doctoral appointments at the New York University and at the University of New South Wales in Sydney he applied label-free and label-based approaches to study biomarkers, protein-protein interactions and PTMs. In 2013 Pawel joined Queensland University of Technology in Brisbane to lead Proteomics and Small Molecule Mass Spectrometry Core in the QUT Central Analytical Research Facility. He supervises research that utilizes LCMS and GCMS instrumentation. Pawel’s interest lays in leveraging the power of recent advancements in data acquisition strategies for MS-based proteomics to drive clinical veterinary research into the next generation. More recently, he has started using quantitative mass spectrometry to study metabolites and other small molecules.

Teaching Old Dog New Tricks: Adaptation of Skyline to Analyze Untargeted Metabolomics Data Collected on GCMS Instrument

Processing untargeted metabolomics data collected on GCMS instruments comes with specific challenges and often requires specialized (or expensive) data analysis software. Moreover, metabolomics researchers tend to apply laboratory-specific GCMS data processing criteria depending on the functionalities available in their favorite software package which prevents others from being able to reproduce the results if they do not have access to the same tools. Read More
Having used Skyline for proteomics data analysis since its early days, we decided to leverage of its recent support for small molecules, and we have developed GCMS data analysis pipeline that utilizes identical criteria as for proteomics datasets and thus effectively we have standardized our quantitative mass spectrometry workflows across majority of our MS platforms and datasets. This Skyline-based pipeline has been subsequently deployed in another mass spectrometry facility in Queensland with positive feedback from its users. In our recent work we have successfully applied our new pipeline to understand the mechanism of sexual selection of fruit flies through GCMS-based metabolomics.


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Birgit Schilling


Birgit Schilling Birgit Schilling, Ph.D. is assistant professor and the director of the Mass Spectrometry Core at the Buck Institute for Research on Aging. She is particularly interested in protein chemistry and modern mass spectrometric technologies to advance the field of proteomics mainly in the context of aging research. Her several research projects include investigation of protein phosphorylation, glycosylation, acetylation, and other posttranslational modifications. We routinely perform quantitative proteomics studies investigating differential expression of intracellular proteins as well as analyzing protein secretomes during disease and aging processes. My group has adopted several novel proteomic technologies and are using proteomic data-independent acquisitions (DIA), or SWATH which allows us to accurately determine changes in relative protein expression level between multiple different conditions. In addition, she has intensively used label-free protein quantification approaches (MS1 Filtering), as well as newer quantitative and highly comprehensive workflows, such as high-resolution data-independent acquisitions (DIA or SWATH Quantification), and parallel reaction monitoring (PRM or high resolution MRM).

Skyline: 10-year Retrospective

Ten years ago a group of about 20 dedicated triple quadrupole operators met at the Broad Institute to learn about the features of Skyline -- this modest beginning evolved into the Skyline User Group Meeting -- the reason you are here today. Nowadays, the Skyline user base has grown significantly with 94,000 new installations of Skyline since 2009. Sue and I will provide an entertaining retrospective over the last 10 years – what were its key achievements? Read More
How did Skyline bring results from labs together even when they used different instrumentation? By keeping its format, vendor-neutral, Skyline has developed great support from all the instrument vendors. In the initial days, we used Skyline for SRM supporting CPTAC projects, system suitability and biomarker research. Over time, the development team added new scan-type capabilities, such as MS1-Filtering, PRM and DIA and now even support for small molecule workflows! Great new features were implemented including iRT (indexed retention time), tool store connections and even linking Skyline with Panorama – the online repository for Skyline research data! A truly comprehensive quantitative environment for everybody, everywhere in the world!


[PDF





Tobias Schmidt


Tobias Schmidt Tobias Schmidt is a fourth-year Ph.D. candidate at the chair of Proteomics and Bioanalytics of Prof. Kuster in Freising, Technical University Munich, Germany. Before coming to the Technical University of Munich for his MSc in molecular biotechnology he spent three years at the Karlsruhe Institute of Technology studying mathematics. His research interest is in in-memory databases (ProteomicsDB), combining modern machine learning with high quality synthetic and real-world data as well as porting legacy (academic) systems to new technologies. His doctoral thesis work explores among other things the usage of his prediction tools for data analysis pipelines requiring high-quality spectral libraries.

Using Prosit for PRM assay development and optimization

Setting up and analyzing MRM/PRM assays require prior information on the retention times and fragmentation spectra of peptides. For so far unobserved peptides, synthetic peptides can be used to obtain these characteristics, however, are costly with uncertain results. We propose a novel cost-efficient approach which utilized our deep learning framework Prosit for the generation of in-silico spectral libraries with near reference data quality for virtually any peptide on a proteome-wide scale. Read More
Skyline-compatible libraries can be generated via ProteomicsDB on-demand and thus allow an initial screening of any peptide of interest. We demonstrate this approach on a dataset which was successfully acquired and analyzed using a predicted library. Subsequently, detected peptides were validated using synthetic peptides. Furthermore, existing libraries can be optimized by using Prosit’s CE-dependent predictions to weaken or boost specific fragments. Thus, predicted libraries supplement prior information and enable the investigation of unobserved proteins and peptides.


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Selene Swanson


speaker Selene Swanson's professional career started in the late 80’s at the Microchemical Facility, University of Minnesota as a junior scientist primarily responsible for protein sequencing, peptide synthesis, DNA sequencing/synthesis and amino acid analysis. After a journey through oncology, hematology, gene therapy and nephrology, she landed in the field of proteomics in the early 2000’s at the Proteomics Center of the Stowers Institute for Medical Research. In 2016, after learning the ropes in MudPIT, she was asked to explore the small molecules arena, specifically modified ribonucleosides, given the availability of state-of-the-art mass spectrometers in the center. With the limited open resources available for analyzing PRM data from small molecules, Skyline has proven to be an invaluable resource to meet our needs.

Absolute quantitative analysis of modified ribonucleosides in tRNA and mRNA using Skyline

Epitranscriptomics is a link between epigenomics and proteomics. Understanding the role of RNA modifications is important in elucidating many fundamental cellular processes such as RNA structural stability, splicing, and translation efficiency. Using a Lumos™ Tribrid™ Mass Spectrometer and Skyline, modified ribonucleosides (RNs) in E. coli tRNA and mammalian mRNA were identified and quantified based on calibration curves generated from known standard RNs. The detection levels of the four 2-O’-methylated RNs ranged from undetectable to 0.7ng and 1.8ng out of 500ng each for mammalian mRNA and tRNA, respectively. Read More
For m7G, an evolutionarily conserved modification of eukaryotic mRNA, 0.46ng was detected from 500ng of hydrolyzed mammalian mRNA. 5’ m7G plays an important role in the life cycle of mRNA such as pre-mRNA processing, nuclear export, and cap-dependent protein synthesis. The ability to identify and quantify these ribonucleoside modifications fosters a mean to compare their abundance level in various cellular and disease states


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Sebastian Vaca


Sebastian Vaca Sebastian Vaca , Ph.D. received his Master’s degree in Analytical Sciences from the University of Strasbourg, France. Afterward, he joined the BioOrganic Mass Spectrometry Laboratory (LSMBO, Strasbourg) to work with Dr. Alain Van Dorsselaer and Dr. Christine Carapito. His research aimed at improving the proteome characterization by quantitative mass spectrometry and proteogenomics method development. He received his Ph.D. in June 2016 and joined Dr. Jacob Jaffe’s lab at The Broad Institute, Cambridge, MA. He focuses on Data-Independent Acquisition method development and data analysis in order to understand phosphosignaling in cancerous cells. Sebastian’s motivation to develop an automated data-curation tool came from the hundreds of hours of sitting in front of a computer manually integrating chromatographic peaks.

Avant-garde: A Skyline External Tool for automated data-driven DIA data curation

Developments in Data-independent Acquisition data analysis have enabled the detection of large numbers of peptides. However, most tools focus on statistical validation of peptide detection (using target/decoy approaches) but do not address the quantitative suitability of the signals extracted. In practice, time-consuming manual signal curation is still required for rigorous quantitation. Here we introduce Avant-Garde (AvG), a Skyline External Tool for automated MS data curation meant to polish the results of DIA/PRM analysis tools and assess the quantitative suitability of DIA signals. Read More
AvG uses three modules to refine DIA data: 1) A transition refinement module eliminates interferences and noise. 2) A peak boundary refinement module improves peak picking without the need for RT standard peptides. 3) A peak scoring module estimates the false discovery rate (FDR) for quantitative suitability using intuitive metrics. AvG was made possible by Skyline’s Annotations feature that allows the import of external data.


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