Use a Bigger SWATH Library and Get More Protein Coverage From Your Sample

Mar 18, 2016 | Blogs, Life Science Research | 0 comments


Calling all SWATH® Acquisition users!

If you have a TripleTOF® System and you’re using SWATH Acquisition for your quantitative proteomics experiments, you’ve made the right choice. SWATH is a data independent workflow that enables data to be acquired for every detectable analyte in a complex sample. You create a digital map of the sample that can be mined for new information any time new biological insights are hypothesized. It’s all there!

Since the introduction of the workflow at HUPO 2010 in Sydney, Australia, we have been working to further improve the workflow. Optimizations in data acquisition such as smaller and variable Q1 window widths and increased instrument dynamic range have provided increased depth of coverage for your proteomics sample, without compromising reproducibility or data quality. 

But what about data processing? Can we improve even further?

The answer is yes! Typically an ion library is used for targeted processing of SWATH data for peptide and protein identification and quantitation. The ion library contains the masses of the peptide ions, sequence-specific fragment ions, relative fragment intensities, and relative retention times, and is easily generated by performing a simple data-dependent acquisition and database search.

In this study, scientists from SCIEX demonstrate that for biological systems that will be studied repeatedly, it is worth taking the extra time to create a deeper ion library as much more information can be extracted from the SWATH acquisition data.

For example, SWATH replicates of a HEK human cell lysate were acquired and processed using three different libraries:

  1. A simple 1D human cell line library generated using traditional data-dependent acquisition strategy (IDA)
  2. A 2D human cell line library using a more in-depth 2D off-line fractionation/IDA strategy
  3. Pan-Human library (PHL) library using a large number of different human cell lines extensive fractionation and IDA strategy 

Figure 1. The Impact of Deeper Ion Libraries on Extraction of Quantitative Data from Human Cell Lysate SWATH® Acquisition Data. 

As shown in Figure 1, a 118% gain in quantified proteins was observed using a simple ion library (1D HEK) and an extensive ion library (PHL). And the quality and reproducibility of the quantitation are maintained even into the low abundant protein/peptide regime.

A researcher can balance the library generation time with the depth of coverage needed. The three libraries outlined here took increasingly more time to generate. A simple IDA experiment can take only a matter of hours. A 2D fractionation followed by LC-MS/MS on each fraction can be performed in a matter of days. And some groups have invested significantly more time in library generation using multiple cell types and a large degree of fractionation to cover as much of the proteome as possible.

This work also highlights the superior dynamic range of quantitative information that is present in a SWATH Acquisition data file over the traditional data dependent approaches for quantitative proteomics. To read more details of this work, and to see what size

To read more details of this work, and to see what size ion library would make the most sense for your biological system of study, download the full technical note.


ProteinPilot phosphopeptide library and DIA-NN

I have prepared a spectral library for a phosphopeptide enriched sample and I am generating my SWATH samples from similarly enriched samples. The problem is that when I use DIA-NN for the retention time alignment and quant, it doesn’t recognise the terminology of the spectral library annotated by ProteinPilot. DIA-NN recognises Unimod:21 for phosphorylation, but PP uses phospho(Tyr) etc. Other than changing the data dictionary to get around the mismatch, anyone have any suggestions for how I might resolve this? Thank you, Roz

Current proteomics software compatibility for ZenoTOF 7600 system

Below is a summary of various other software packages that are useful for processing proteomics data from the ZenoTOF 7600 system.  Note this list is not comprehensive and only covers the tools we have lightly tested to date. Acquisition Type Software Files needed...

Easy switching of sources and LC flow regimes on the ZenoTOF 7600 system

This series of videos outlines how a user can easily switch sources and operate the system in different LC flow regimes.

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Christie Hunter is the Director of Applications at SCIEX. Christie has worked at SCIEX for 20 years, pioneering many workflows in quantitative proteomics. Christie was an early user of SWATH acquisition and played a big role in evolving the workflows and driving adoption of this new data independent approach with many proteomic researchers. Christie and her team are focused on developing and testing innovative MS workflows to analyze biomolecules, and work collaboratively with the instrument, chemistry and software research groups.



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