Skyline Software for Peptide MRM Method Development

Skyline Software

For research use only. Not for use in diagnostic procedures.

Skyline software is a Windows client application for building multiple reaction monitoring (MRM) and full-scan (MS1 and MS/MS) quantitative methods and analyzing the resulting mass spectrometer data. It employs cutting-edge technologies for creating and iteratively refining targeted methods for large-scale proteomics studies.

Download Skyline software, free of charge, from the MacCoss Lab at the University of Washington.

Skyline is a comprehensive package for building and optimizing multiple reaction monitoring (MRM) assays. Proteins and peptides can be loaded into Skyline from previous results or pasted from Microsoft Excel or from library data. Then, using predictive algorithms, the best charge state and fragment ions for each peptide are chosen for the acquisition method. The user has complete control over peptide settings and transition settings and can specify the number of transitions per method, a standard or Scheduled MRM™ Assay method, set up parameter optimization, or browse to a template method that can have all chromatographic settings included.

A rich array of graphs displays results and provides powerful tools for inspecting data integrity during acquisition, helping instrument operators to identify problems prompty. With sufficient screen resolution, Skyline can display useful information in 10 or more graph panes simultaneously, providing updates as the selection changes without noticeable delays.

Figure 1: Exploring MRM data generated using the QTRAP System. Peptides and transitions are displayed at the left. Clicking on a peptide and transition from the navigation pane shows the corresponding MRM transition and MS/MS data. Dots next to peptides and transitions indicate signal strength. Green indicates a strong ion signal, while yellow indicates decreasing signal strength. No dot indicates there was no acquisition for that peptide transition.

Figure 2: Exploring high-resolution MRM (MRM^HR) data generated using the TripleTOF System. High-resolution extracted ion chromatorgrams (XICs) of precursors and fragments are generated for each peptide and displayed. In this case, the chromatographic retention times and intensities for each peptide and fragment ion are displayed along with other fragment ions for the same peptide, enhancing identification of optimum fragments along with potential problem fragments.

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