Expansion of the Ion Library for Mining SWATH-MS Data
through Fractionation Proteomics
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Abstract
The strategy of sequential window
acquisition of all theoretical
fragment ion spectra (SWATH) is emerging in the field of label-free
proteomics. A critical consideration for the processing of SWATH data
is the quality of the ion library (or mass spectrometric reference
map). As the availability of open spectral libraries that can be used
to process SWATH data is limited, most users currently create their
libraries in-house. Herein, we propose an approach to construct an
expanded ion library using the data-dependent acquisition (DDA) data
generated by fractionation proteomics. We identified three critical
elements for achieving a satisfactory ion library during the iterative
process of our ion library expansion, including a correction of the
retention times (RTs) gained from fractionation proteomics, appropriate
integrations of the fractionated proteomics into an ion library, and
assessments of the impact of the expanded ion libraries to data mining
in SWATH. Using a bacterial lysate as an evaluation material, we employed
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
to fractionate the lysate proteins and constructed the expanded ion
library using the fractionation proteomics data. Compared with the
ion library built from the unfractionated proteomics, approximately
20% more peptides were extracted from the expanded ion library. The
extracted peptides, moreover, were acceptable for further quantitative
analysis