Sunday, 6 April 2014

SWATH-MS and next-generation targeted proteomics

For proteomics, two main LC-MS/MS strategies have been used thus far. They have in common that the sample proteins are converted by proteolysis into peptides, which are then separated by (capillary) liquid chromatography. They differ in the mass spectrometric method used.

The first and most widely used strategy is known as shotgun proteomics or discovery proteomics. For this method, the MS instrument is operated in data-dependent acquisition (DDA) mode, where fragment ion (MS2) spectra for selected precursor ions detectable in a survey (MS1) scan are generated (Figure 1 - Discovery workflow). The resulting fragment ion spectra are then assigned to their corresponding peptide sequences by sequence database searching (See Open source libraries and frameworks for mass spectrometry based proteomics: A developer's perspective).

The second main strategy is referred to as targeted proteomics. There, the MS instrument is operated in selected reaction monitoring (SRM) (also called multiple reaction monitoring) mode (Figure 1 - Targeted Workflow). With this method, a sample is queried for the presence and quantity of a limited set of peptides that have to be specified prior to data acquisition. SRM does not require the explicit detection of the targeted precursors but proceeds by the acquisition, sequentially across the LC retention time domain, of predefined pairs of precursor and product ion masses, called transitions, several of which constitute a definitive assay for the detection of a peptide in a complex sample (See Targeted proteomics) .

Figure 1 - Discovery and Targeted proteomics workflows

Both methods have different and largely complementary preferred uses and performance profiles. Specifically, shotgun proteomics is the method of choice for discovering the maximal number of proteins from one or a few samples. In contrast, targeted proteomics is well suited for the reproducible detection and accurate quantification of sets of specific proteins in many samples as is the case in biomarker or systems biology studies.

At present, however, SRM is limited to the measurements of a few thousands transitions per LC-MS/MS run. To alleviate the limitations of the method, strategies have been developed that rely on neither detection nor knowledge of the precursor ions to trigger acquisition of fragment ion spectra. Those methods operate via unbiased “data-independent acquisition” (DIA), in the cyclic recording, throughout the LC time range, of consecutive survey scans and fragment ion spectra for all the precursors contained in predetermined isolation windows. Using such scans, the link between the fragment ions and the precursors from which they originate is lost, complicating the analysis of the acquired data sets. To date, the composite spectra generated by DIA methods have been principally analyzed with the standard database searching tools developed for DDA, either by searching the composite MS2 spectra directly or by searching pseudo MS2 spectra reconstituted postacquisition based on the co-elution profiles of precursor ions (from the survey scans) and of their potentially corresponding fragment ions.

"SWATH MS" is an alternative approach to proteome quantification that combines a high specificity DIA method with a novel targeted data extraction strategy to mine the resulting fragment ion data sets. The method uniquely combines a DIA methods with a innovative data analysis approach based on targeted data extraction developed in the Aebersold lab. Like in other DIA methods, the mass spectrometer cycles through precursor acquisition windows designed to cover the whole range of 400-1200 m/z - in which most of the proteotypic peptide precursors of an organism fall - within 2-4 seconds. During each cycle, the mass spectrometer will fragment all precursors from a given precursors window (e.g. 475 - 500 m/z for 25 Da windows) and record a complete, high accuracy fragment ion spectrum. The same range will be fragmented again in the next cycle, thus providing a time-resolved recording of fragment ions that elute on the chromatography. Thus the SWATH method provides highly multiplexed fragment ion spectra that are deterministically recorded over the complete chromatographic time.  The SWATH MS data analysis workflow consists of using a targeted data extraction strategy to query the acquired fragment ion maps for the presence and quantity of specific peptides of interest, using a priori information contained in spectral libraries.

The combination of high specificity fragment ion maps and targeted data analysis using information from spectral libraries of complete organisms offers unprecedented possibilities for the qualitative and quantitative probing of proteomes. 

If you are interested in the topic you can also read: 

- Gillet LC, Navarro P, Tate S, Röst H, Selevsek N, Reiter L, Bonner R, Aebersold R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics. 2012 Jun;11(6):O111.016717. Epub 2012 Jan 18. PMID 22261725

 - Panchaud, A., A. Scherl, S. A. Shaffer, P. D. von Haller, H. D. Kulasekara, S. I. Miller, and D. R. Goodlett (2009, August). Precursor acquisition independent from ion count: how to dive deeper into the proteomics ocean. Analytical chemistry 81 (15), 6481-6488.

- OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data Nat Biotech, Vol. 32, No. 3. (10 March 2014), pp. 219-223, doi:10.1038/nbt.2841 by Hannes L. Rost, George Rosenberger, Pedro Navarro, et al.