April 29, 9:00AM-May 1, 12:30PM : Targeted proteomics with Skyline
Description
This 2.5 day short course will walk through the process of designing, developing, and statistically evaluating LC-MS methods for the targeted quantification of peptides with high confidence. Topics covered will include peptide selection from target proteins, optimization of peptide transitions, LC gradient, collision energy, and discussion on internal standards. Attendees will learn rigorous method evaluation and performance characterization, limits of detection and quantification calculation, and use of routine System Suitability QC. Case studies and tutorials will be provided for hands-on participation with open-source software Skyline (https://skyline.ms/project/home/software/Skyline/begin.view).
Target audience
- Target audience are experimental scientists with experience in proteomic research. We also welcome computational scientists interested in working with raw mass spectrometric data.
Speakers
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- Sue Abbatiello, Andy Hoofnagle, Christina Ludwig, Lindsay Pino
Tentative schedule
Monday, April 29, 2019
- 8:00 a.m. Registration
- 9:00 a.m. Keynote: Introduction to quantitative proteomics and lessons from CPTAC, Susan Abbatiello
- 10:00 a.m. Lecture: Introduction to targeted and chromatography-based proteomics, Christina Ludwig
- 10:30 a.m. Refreshments
- 11:00 a.m. Lecture cont’n: Introduction to targeted and chromatography-based proteomics, Christina Ludwig
- 11:30 a.m. Lecture: Introduction to SKYLINE, Christina Ludwig
- 12:00 a.m. Hands-on: Setting up a Skyline document, developing a targeted list, refining transitions, optimizing collision energy, Susan Abbatiello
- 12:30 p.m. Lunch
- 1:30 p.m. Hands-on cont’n: Setting up a Skyline document, developing a targeted list, refining transitions, optimizing collision energy, Susan Abbatiello
- 2:00 p.m. Lecture: iRT, retention time, standards, Lindsay Pino
- 2:30 p.m. Hands-on: iRT calculator, Lindsay Pino/Christina Ludwig
- 3:00 p.m. Refreshments
- 3:30 p.m. Hands-on cont’n: iRT calculator, Lindsay Pino/Christina Ludwig
- 4:00 p.m. Hands-on: Plasma dataset, Lindsay Pino
- 5:00 p.m. Q&A and adjourn
Tuesday, April 30, 2019
- 9:00 a.m. Keynote: HDL Proteomics in Chronic Kidney Disease, Andy Hoofnagle
- 10:30 a.m. Refreshments
- 11:00 a.m. Lecture: Standards and validation experiments, Lindsay Pino
- 11:30 a.m. Hands-on: Peptide stability+reproducibility+repeatability experiments, Lindsay Pino/Christina Ludwig
- 12:30 p.m. Lunch
- 1:30 p.m. Lecture: Absolute quantification and calibration curves, Susan Abbatiello
- 2:15 p.m. Hands-on: Single/multi-point calibration in Skyline, Lindsay Pino
- 3:00 p.m. Refreshments
- 3:30 p.m. Hands-on: Determining LOD/LOQ from calibration curve data, Lindsay Pino
- 4:15 p.m. Lecture: PTMs and phospho-site localization in targeted proteomics, Christina Ludwig
- 5:00 p.m. Q&A
- 6:00 p.m. Dinner at Back Bay Social dinners
Wednesday, May 1, 2019
- 9:00 a.m. Lecture: System suitability, QC design, Susan Abbatiello
- 10:30 a.m. Refreshments
- 11:00 a.m. Hands-on: Panorama and PanoramaQC, Lindsay Pino
- 12:30 p.m. Wrap-up and Kahoot!, Christina Ludwig