Potential useful information to review and add to the readthedocs page - from learning unit 6

[timosachsenberg]

Targeted proteomics
Targeted proteomics/metabolomics is based on a list of known analytes (proteins, metabolites). Targeted methods are in contrast to so-­called discovery mode or shotgun proteomics, where proteins/metabolites are identified and quantified as comprehensively, as possible.
SRM/MRM
Selected Reaction Monitoring (SRM) and Multiple Reaction Monitoring (MRM) use the signal of selected MS2 fragment ions for quantification. It is typically performed on triple-quadrupole instruments: Q1 selects a peptide ion, Q2 fragments the peptide, and Q3 selects a specific fragment ion for the detector, as shown below.

The double mass selection reduces possible interferences between ions, quantification through MRM signal area.

SRM vs. MRM
SRM is monitoring only a single fixed mass window, while MRM scans rapidly over multiple (very narrow) mass windows and thus acquires traces of multiple fragment ion masses in parallel. So MRM is the application of SRM to multiple product ions from one or more precursor ions. Following graph is an example of MRM, multiple scans (only two are listed as an example) are recoded in parallel.

Advantages of SRM/MRM (compared to shotgun)
Better sensitivity
Better linear range (4-5 orders of magnitude)

Structure of SRM data and definition of terms(Reiter et. al., 2011): a) REpresentation of the SRM measurement of one peptide. b): Peak group features taht can be used to identify a true peak group. Red indicates an unexpected behavior for true peak groups.

The data resulting from the measurement of one transition or transition group are called a trace or transition group record, respectively. In one transition group record, several peak groups can be identified that potentially represent the peptide of interest. If a peak group (as illustrated in above figure) is derived from the targeted peptide, the peaks tend to have similar rentention time profiel and shape. Further, the relative intensities of the fragment ions must correspond to previously measured intensity ratios. If a reference peptide is in the sample, the relative intensities for all corresponding traces as well as peak shape and elution time should be similar for intrinsic peptide and reference.

Computational challenges
MRM Assay consists of a transition list of SRM. For each SRM transition, the expected retention time, precursor ion m/z, and fragment ion m/z need to be specified for the assay. Transition list is uploaded to the instrument prior to the analysis and controls.

Assay construction process leads to the main challenge for computation and analysis. The construction is to determine a transition list given a list of proteins. It can be based either on experimentally determined tandem spectra (to identify the most intense fragment ions) or on predicted spectra. Assays need also to be optimized to avoid interferences and to optimize instrument settings for each transition. There already exists some automated assay analysis: given an assay, we want to automatically quantify a sample. To do so, some tools will be introduced later.

Overview SWATH-MS
SWATH-MS (Sequential Windowed data independent Acquisition of the Total High-resolution Mass Spectra) is an untargeted and data independent acquisition strategy for mass spectrometers. It can be compared to the MRM strategy when going from multiple targeted peptides to all peptides in the sample. Although the speed of MS instruments increased in recent years, this strategy is only feasible by increasing the narrow precursor windows (like in MRM) to bigger sequential windows covering a complete, fixed m/z range (usually 400-1200 Th) in a small cycle time of a few seconds. This means, in each cycle the full range is chopped into windows (of around 25 Th) and fragment scans are generated for them, resulting in a complete fragment library of the whole sample in a very small period of retention time. Since in every following cycle another fragment scan is initiated for the same windows, we can analyze the course of the intensities of the fragments in each window over time (like in SRM/MRM).
However, due to the large m/z windows used for fragmentation, we get heavily multiplexed fragment scans and the database search approach (used in shotgun proteomics) is not applicable anymore. Another problem that arises from allowing several fragments to be measured in the MS2 scans is the interference of these ions. For adapted algorithms, see the following chapters.
To illustrate some of the main differences between SRM/MRM, Shotgun (exploratory/discovery) and SWATH acquisition, we present the following Figures. The first Figure shows the same MS1 map of an experiment and marks the precursor windows (red arrows) chosen for fragmentation scans for two different strategies (Shotgun left, MRM right). You can see the data dependence of the shotgun method as only features with high intensities are picked for MS2 scans. In MRM, two precursor windows were chosen to be surveyed over the whole RT span. In each MS2 scan of the MRM approach, different fragment ions (seven colored bullets in the MS2 inlets on the right) can be determined and their XICs (Extracted Ion Chromatograms; middle right inlet) can be plotted.

For SWATH (Figure below), the windows are aligned in a sequential order and cover a range of usually 25 Th (instead of less than 1 Th in SRM/MRM and Shotgun strategies). To cover a preset range of 400-1200 Th for example, we need (1200 - 400)/25 = 32 windows (so called swaths). If a fragmentation scan then needs 0.1s, the time for a full cycle is 3.2s. This means, we get a fragmentation scan of a certain window every 3.2 seconds and we can generate a smooth profile of the fragments over RT.

In scientific literature, SWATH acquisition was first described by Gillet et al.

Heavily multiplexed fragment spectra
As described in the chapter before, a mass spectrometer run in SWATH acquisition mode, will produce MS2/fragment scans of 25 Th windows. These windows most certainly include fragments of multiple precursors, such that a database search like in shotgun methods is not feasible. Also for MRM-like analysis with e.g. mProphet, after extracting the XICs of the fragments over all MS2 scans along RT, lots of interfering traces will be found.
This complicates the search for peak groups associated with a target precursor in the XICs (see MRM, LU 6AB) and raises the need for a sound statistical evaluation. Thankfully, the fact that SWATH acquisition generates MS2 spectra at every time point, enables one to extend the "chromatographic" scores used in mProphet by additional "spectral" scores. This idea is used in the OpenSWATH library integrated into OpenMS.

OpenSwath
OpenSWATH (Roest et al.) is an open-source (Modified BSD License) software that allows targeted analysis of DIA data in an automated, high-throughput fashion. OpenSWATH is cross-platform software, written in C++, that relies only on open data formats, allowing it to analyze DIA data from multiple instrument vendors. It is fully integrated into and distributed together with OpenMS .

The classes, i.e. the steps of this pipeline will be explained on the following pages. A graphical overview of the steps is given in the next Figure.
Subfigure a) shows the overall acquisition strategy again plus the result on MS2 level for a specific swath window as inlet. Subfigure b) then
sketches the steps performed by the OpenSWATH algorithms. Please note that although the inlet showing the transitions on the MS2 level (middle left)
uses the map from above, its axes are switched (probably erroneously).

Sketch of the steps involved in the analysis done by OpenSWATH.