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Abstract
<p class="first" id="P1">Quantitative analysis of proteomes across multiple time points,
organelles, and perturbations
is essential for understanding both fundamental biology and disease states. The development
of isobaric tags (e.g., TMT) has enabled the simultaneous measurement of peptide abundances
across several different conditions. These multiplexed approaches are promising in
principle because of advantages in throughput and measurement quality. However, in
practice, existing multiplexing approaches suffer from key limitations. In its simple
implementation (TMT-MS2), measurements are distorted by chemical noise leading to
poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this but
requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter
ion approach (TMTc) produces high accuracy measurements and is compatible with many
more instruments, like quadrupole-Orbitraps. However, the required deconvolution of
the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which
adds the modeling of the MS2-isolation step into the deconvolution algorithm. The
resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty
cycle and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and
comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able
to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare
TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+
quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time, the median
coefficient of variation improves from 13% to 4%. Thus, TMTc+ advances quantitative
proteomics by enabling accurate, sensitive, and precise multiplexed experiments on
more commonly used instruments.
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[1
]Department of Molecular Biology and the Lewis-Sigler Institute for Integrative Genomics,
Princeton University, Princeton, New Jersey 08544, United States