4 research outputs found
Recommended from our members
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
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
Accurate Multiplexed Proteomics at the MS2 Level Using the Complement Reporter Ion Cluster
Isobaric labeling strategies, such as isobaric tags for
relative
and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have
promised to dramatically increase the power of quantitative proteomics.
However, when applied to complex mixtures, both the accuracy and precision
are undermined by interfering peptide ions that coisolate and cofragment
with the target peptide. Additional gas-phase isolation steps, such
as proton-transfer ion–ion reactions (PTR) or higher-order
MS3 scans, can almost completely eliminate this problem. Unfortunately,
these methods come at the expense of decreased acquisition speed and
sensitivity. Here we present a method that allows accurate quantification
of TMT-labeled peptides at the MS2 level without additional ion purification.
Quantification is based on the fragment ion cluster that carries most
of the TMT mass balance. In contrast to the use of low <i>m</i>/<i>z</i> reporter ions, the localization of these complement
TMT (TMT<sup>C</sup>) ions in the spectrum is precursor-specific;
coeluting peptides do not generally affect the measurement of the
TMT<sup>C</sup> ion cluster of interest. Unlike the PTR or MS3 strategies,
this method can be implemented on a wide range of high-resolution
mass spectrometers like the quadrupole Orbitrap instruments (QExactive).
A current limitation of the method is that the efficiency of TMT<sup>C</sup> ion formation is affected by both peptide sequence and peptide
ion charge state; we discuss potential routes to overcome this problem.
Finally, we show that the complement reporter ion approach allows
parallelization of multiplexed quantification and therefore holds
the potential to multiply the number of distinct peptides that can
be quantified in a given time frame
Generation of Multiple Reporter Ions from a Single Isobaric Reagent Increases Multiplexing Capacity for Quantitative Proteomics
Isobaric labeling strategies for
mass spectrometry-based proteomics
enable multiplexed simultaneous quantification of samples and therefore
substantially increase the sample throughput in proteomics. However,
despite these benefits, current limits to multiplexing capacity are
prohibitive for large sample sizes and impose limitations on experimental
design. Here, we introduce a novel mechanism for increasing the multiplexing
density of isobaric reagents. We present Combinatorial Isobaric Mass
Tags (CMTs), an isobaric labeling architecture with the unique ability
to generate multiple series of reporter ions simultaneously. We demonstrate
that utilization of multiple reporter ion series improves multiplexing
capacity of CMT with respect to a commercially available isobaric
labeling reagent with preserved quantitative accuracy and depth of
coverage in complex mixtures. We provide a blueprint for the realization
of 16-plex reagents with 1 Da spacing between reporter ions and up
to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent.
We anticipate that this improvement in multiplexing capacity will
further advance the application of quantitative proteomics, particularly
in high-throughput screening assays