Christie Hunter


What is the best way to build spectral ion libraries for use in processing of proteomics SWATH acquisition data?

The information needed for targeted data extraction in our current SWATH acquisition workflow is relatively simple.  You need the parent ion m/z; you need the m/z and relative intensities of the major fragment ions that are produced during MS/MS; and you need to know the relative retention times of the peptides of interest. Currently researchers are employing two strategies: a data-driven approach per studty or a pan-proteome / repository approach.

In the data-driven approach, extensive data- or information-dependent analysis (IDA) is done on the samples of interest, and proteins and peptides are identified from this acquisition using ProteinPilot software or other database search tools. The identified peptides in the search result now serve as an ion library to apply to the SWATH acquisition runs for this study.  The library generation acquisition can be done with 1D LC-MS or 2D LC-MS depending on the depth of library required for the study.

>Spectral library generation for SWATH acquisition in less than 20 hours, SCIEX technical note RUO-MKT-02-12767-A.

In the repository approach, MS/MS data from public or internal repositories are mined for target proteins and peptides and used to create the ion library. Or samples from many sources that provide a very broad coverage of the whole proteome are extensively analyzed to produce a deep library.  Using libraries generated in this way will typically require retention time calibration strategies as often the retention times in the current study will be different.

Researchers from Dr. Ruedi Aebersold’s laboratory at ETH Zurich and Dr Robert Moritz’s lab at ISB Seattle have been working on developing whole proteome libraries for use in SWATH acquisition studies. They are targeting the widely used proteomes that will be of very good use to the research community.  The first of such ion libraries (M. tuberculosis ) has been made available on the ISB website (   Other libraries such as human2 and yeast3 are also available.

  1. Enabling systems biology driven proteome wide quantification of Mycobacterium tuberculosis, SCIEX technical notes RUO-MKT-02-4292-C.
  2. Rosenberger G et al. (2014) A repository of assays to quantify 10,000 human proteins by SWATH MS. Scientific Data, 1, 140031.
  3. Selevsek N et (2015) Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH mass spectrometry. Mol Cell Prot. 14, 739-749.


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