Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster

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.