When it comes to testing complex cellular samples, SWATH® Acquisition in combination with deeper ion libraries provides more reproducibly quantified proteins. Learn how to balance library generation with results required from SCIEX experts.

1. 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

2. 2 © 2016 AB Sciex Mini Slide Deck
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