Date post: | 20-Jan-2017 |
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Extending the Depth of Coverage in SWATH® Acquisition with Deeper Ion Libraries Data Independent Acquisition on the TripleTOF® 6600 Systems
RUO-MKT-11-3675-A For Research Use Only. Not for use in Diagnostic Procedures
Mini Slide Deck
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Extending Depth of Coverage
How can we extend the depth of coverage for a highly complex sample?
1 Improved data Quality Using Q1 Variable Window Widths in SWATH Acquisition, SCIEX Technical note RUO-MKT-02-2879-A
2 Increasing Depth of Coverage in Data Independent Acquisition with high Sample Loads and Smaller Q1 windows, SCIEX Technical note RUO-MKT-02-3245-A
• Increase specificity during data acquisition by decreasing chemical noise/interferences1
• Smaller Q1 window• Variable windows• Longer gradients
• Increase signal during data acquisition2 • Load more sample• MS linear dynamic range
• Deeper ion libraries for data processing• Orthogonal fractionation strategies• Large whole proteome libraries
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Extraction of Quantitative SWATH® Data from Human Cell Lysate
• SWATH replicates from HEK cell lysate were processed with increasingly larger libraries to assess impact on depth of coverage‒ Considering proteins quantified with
<1% peptide FDR and < 20%CV
• 1D HEK library - 2714 proteins
• 2D K562 library - 65% gain in quantified proteins over 1D from same SWATH data
• Pan Human Library PHL1 - significant gain in quantified proteins (118%) over 1D experiment, for a total of 5922 proteins reliably quantified
Impact of Deeper Ion Libraries
For Research Use Only. Not for use in Diagnostic Procedures
1 Rosenberger G et al. (2014) Scientific Data, 1, 140031.
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Balancing Library Depth with Library Generation Time
• Larger ion libraries allow more proteins / peptides to be reproducibly quantified, but need more time to be generated
• Based on the library depth needed, the generation time can be balanced by adjusting:‒ # of IDA experiments, # of fractions
generated, number of related cell sources, micro vs nanoflow, etc
• This work highlights:‒ Ion library generation strategy can be
matched to the depth of coverage required for the biology under study and the time needed to create the library
‒ Superior dynamic range of the SWATH technique over traditional data dependent based strategies