Evaluation of a Dual Isolation Width Acquisition Method for Isobaric Labeling Ratio Decompression

Isobaric labeling is a highly precise
approach for protein quantification. However, due to the isolation
interference problem, isobaric tagging suffers from ratio underestimation
at the MS2 level. The use of narrow isolation widths is a rational
approach to alleviate the interference problem; however, this approach
compromises proteome coverage. We reasoned that although a very narrow
isolation window will result in loss of peptide fragment ions, the
reporter ion signals will be retained for a significant portion of
the spectra. On the basis of this assumption, we have designed a dual
isolation width acquisition (DIWA) method, in which each precursor
is first fragmented with HCD using a standard isolation width for
peptide identification and preliminary quantification, followed by
a second MS2 HCD scan using a much narrower isolation width for the
acquisition of quantitative spectra with reduced interference. We
leverage the quantification obtained by the “narrow”
scans to build linear regression models and apply these to decompress
the fold-changes measured at the “standard” scans. We
evaluate the DIWA approach using a nested two species/gene knockout
TMT-6plex experimental design and discuss the perspectives of this
approach.