What is the best way to build spectral ion libraries for SWATH™ Acquisition data processing?
A - 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 or a 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.
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. There are two recent papers describing the generation of large ion libraries for human and yeast.
What tools do SCIEX have for generating combined ion libraries that may have been generated from IDA runs of different gradient length (assuming spiked in iRT standards)? Is there a way to convert the different ProteinPilot group files retention time data to a relative "iRT" using spiked in retention time standards so the group files can be combined to a new library?
Currently, we do not have a solution for combining multiple group files or multiple ion libraries that are acquired using different gradient lengths. However, I definitely agree this is an important tool that is needed to allow us added flexibility in the generation of SWATH libraries. We are investigating ways that we could do this.
I do know that a couple of other SWATH groups have developed tools for doing something similar, aligning the retention times onto a single scale. This is a great example of where we would love to see labs with tools such as this develop applications in the BaseSpace environment to share with the community. This will speed the pace of development and enrich the toolkit for all of us! And SCIEX is eager to assist in this tool development, to help expand the application space.