A Multiplex Fragment-Ion-Based Method for Accurate Proteome Quantification

Multiplex proteome
quantification with high accuracy is urgently
required to achieve a comprehensive understanding of dynamic cellular
and physiological processes. Among the existing quantification strategies,
fragment-ion-based methods can provide highly accurate results, but
the multiplex capacity is limited to 3-plex. Herein, we developed
a multiplex pseudo-isobaric dimethyl labeling (m-pIDL) method to extend
the capacity of the fragment-ion-based method to 6-plex by one-step
dimethyl labeling with several millidalton and dalton mass differences
between precursor ions and enlarging the isolation window of precursor
ions to 10 m/z during data acquisition.
m-pIDL showed high quantification accuracy within the 20-fold dynamic
range. Notably, the ratio compression was 1.13-fold in a benchmark
two-proteome model (5:1 mixed E. coli proteins with
HeLa proteins as interference), indicating that by m-pIDL, the ratio
distortion of isobaric labeling approaches and the approximate 40%
ratio shift of the label-free quantification strategy could be effectively
eliminated. Additionally, m-pIDL did not show ratio variation among
post-translational modifications (CV = 6.66%), which could benefit
the measurement of universal protein properties for proteomic atlases.
We further employed m-pIDL to monitor the time-resolved responses
of the TGF-β-induced epithelial–mesenchymal transition
(EMT) in lung adenocarcinoma A549 cell lines, which facilitated the
finding of new potential regulatory proteins. Therefore, the 6-plex
quantification of m-pIDL with the remarkably high accuracy might create
new prospects for comprehensive proteome analysis.