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

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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