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What is the best strategy for performing retention time calibration when using SWATH® Acquisition?
When processing SWATH® Acquisition data, you want to use a reasonably narrow time window to reduce the chance of incorrect peak integrations. There are currently two strategies being employed today to adjust for differences between the retention times in the spectral ion libraries and the retention times observed in the current sample analysis. Some labs add standard peptides to their samples, both when creating the ion libraries and when running SWATH® Acquisition. This enables the retention times for targeted extractions to be adjusted to correlate with the current acquisition. Retention time alignment can also be done using some of the medium-to-high abundance endogenous peptides present in both the ion library and the current sample. In each case, you want to have confident detections across the whole gradient in order to adjust the retention time scale for the entire sample.
SWATH® Acquisition looks like an exciting technology for targeted quantitative proteomics, but how does it compare to the industry standard MRM technology for quantitative profiling?
Of course, this will depend on which triple quadrupole or QTRAP® system you are comparing to SWATH® Acquisition. We did some work to compare the top end QTRAP 6500 system with SWATH® Acquisition on the TripleTOF® system to understand their relative performance in quantitative profiling. With SWATH® Acquisition, you can quantify very large numbers of peptides and proteins from a single acquisition and achieve quantitative reproducibility that approaches that of MRM analysis. On the QTRAP 6500 system, the numbers of MRMs that can be scheduled are far fewer than that of SWATH® Acquisition, but due to the higher sensitivity of the QTRAP 6500 system and the specificity of MRM, we were able to get better quantitative data on the very low abundant peptides in the samples. One key point is that it is very straightforward to transition from SWATH® Acquisition to MRM on these platforms, since they use the same ion source and collision cell technology; accordingly, all of the assay information can directly transfer.
What are the critical acquisition attributes of a mass spectrometer required for good SWATH® Acquisition data quality?
In data-independent acquisition strategies like SWATH®, an expanded mass isolation window is stepped across a mass range covering the mass-to-charge (m/z) distribution of peptides, and a full scan MS/MS spectrum is collected at each step. High resolution MS/MS is one key attribute, as it enables target fragment ions to be found and extracted with specificity from these complex MS/MS spectra. More importantly, these MS/MS spectra must be acquired at high speed. We find that narrower isolation windows yield higher specificity in data analysis, which means that it is advantageous to take more steps to cover the mass range of your experiment and collect the MS/MS faster to maintain an optimal total cycle time. This is why the TripleTOF® systems are so powerful for this acquisition technique: because high resolution MS/MS can be acquired at very high speed, much smaller isolation windows can be used.
What is the best way to build spectral ion libraries for SWATH® Acquisition data processing?
The information needed for targeted data extraction in our current SWATH® workflow is relatively simple. You need the parent ion m/z, 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 performed on the sample of interest, and proteins and peptides are identified from this acquisition using ProteinPilot™ or other database search tools. The identified peptides in the search result then serve as an ion library to apply to the SWATH® acquisition runs. This interrogation can be accomplished with 1D LCMS or 2D LCMS depending on the depth of library required for the study. In the informatics approach, MS/MS data from public or internal repositories are mined for target proteins and peptides, and are used to create the ion library. Here, retention time calibration strategies should be employed.
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