The lab of Ruedi Aebersold recently published a Nature Protocol for generating peptide libraries for targeted analysis of SWATH MS data-independent acquisition mass spectrometry. This protocol complements a data descriptor (published in Scientific Data by the same authors) for a large-scale human assay library that can be used to support protein quantification by SWATH-MS.
In order to understand an organism’s biology and how it responds to environmental changes (e.g. disease or drug treatments), many researchers would like to be able to correctly identify and even quantify as many of the proteins in any given sample as possible. Mass spectrometry – in particular liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) – is the analytical technique most commonly used for deep and reliable exploration of the proteome.
In bottom-up proteomics, the cells of the sample are lysed, the proteins are digested into shorter peptides, the non-peptide material is removed, and the peptides separated by liquid chromatography. In LC-MS, the column separating the peptides is interfaced with a mass spectrometer and the peptides are analysed as they elute.
Sometimes the researcher will already know which proteins will be most interesting for their study; in these cases Selected Reaction Monitoring (SRM) is a very sensitive mass spectrometry approach to detecting and consistently quantifying the peptides associated with these proteins over many samples. The reactions monitored are the specific fragmentations associated with peptides-of-interest that occur in the mass spectrometer.
In many cases this information will not yet be fully available before the measurement or the number of potential protein-players in the given system might be very large. Data-independent acquisition of mass spectra for proteomics experiments is an alternative approach that extends the number of proteins that can be targeted in a sample from approximately one hundred (in SRM) to several thousand. It is made possible by the development of mass spectrometry instruments that are able to acquire high-resolutioin mass spectra at a very high sampling rate. The analysis method requires the researcher to perform initial experiments to put together a library of time and mass spectrometric coordinates corresponding to the peptides associated with each sample type. These spectra are then used as the basis for the development of highly specific assays to detect and quantify the respective peptide in subsequent samples.
We recently published a protocol for generating peptide libraries for a type of data independent mass spectrometry developed by Ruedi Aebersold and co-workers called SWATH-MS. The assay libraries are generated by collecting high-quality fragment ion spectra in data dependent acquisition mode, and processing these in a number of computational steps as shown in the workflow below.
Scientific Data published a data descriptor for a generic large-scale human assay library to support protein quantification by SWATH-MS. This library was prepared using the procedure described in the Nature Protocol and consists of 1,164,312 transitions identifying 139,449 proteotypic peptides and 10,316 proteins; it was generated by combining the results from 331 measurements of fractions from different cell lines, tissue and affinity enriched protein samples.