Deuterium-Free, Three-Plexed Peptide Diethylation for Highly Accurate Quantitative Proteomics

The deuterium, a frequently used
stable isotope in isotopic labeling
for quantitative proteomics, could deteriorate the accuracy and precision
of proteome quantification owing to the retention time shift of deuterated
peptides from the hydrogenated counterpart. We introduce a novel three-plexed
peptide “diethylation” using only 13C isotopologues
of acetaldehyde and demonstrate that the accuracy and precision of
our method in proteome quantification are significantly superior to
the conventional deuterium-based dimethylation labeling in both a
single-shot and multidimensional LC–MS/MS analysis of the HeLa
proteome. Furthermore, in time-resolved profiling of Xenopus
laevis early embryogenesis, our 3-plexed diethylation
outperformed isobaric labeling approaches in terms of the quantification
accuracy or the number of protein identifications, generating more
than two times more differentially expressed proteins. Our cost-effective
and highly accurate 3-plexed diethylation method could contribute
to various types of quantitative proteomics applications in which
three of multiplexity would be sufficient.