4 research outputs found
Ion Coalescence of Neutron Encoded TMT 10-Plex Reporter Ions
Isobaric
mass tag-based quantitative proteomics strategies such
as iTRAQ and TMT utilize reporter ions in the low mass range of tandem
MS spectra for relative quantification. The recent extension of TMT
multiplexing to 10 conditions has been enabled by utilizing neutron
encoded tags with reporter ion <i>m</i>/<i>z</i> differences of 6 mDa. The baseline resolution of these closely spaced
tags is possible due to the high resolving power of current day mass
spectrometers. In this work we evaluated the performance of the TMT10
isobaric mass tags on the Q Exactive Orbitrap mass spectrometers for
the first time and demonstrated comparable quantification accuracy
and precision to what can be achieved on the Orbitrap Elite mass spectrometers.
However, we discovered, upon analysis of complex proteomics samples
on the Q Exactive Orbitrap mass spectrometers, that the proximate
TMT10 reporter ion pairs become prone to coalescence. The fusion of
the different reporter ion signals into a single measurable entity
has a detrimental effect on peptide and protein quantification. We
established that the main reason for coalescence is the commonly accepted
maximum ion target for MS2 spectra of 1e6 on the Q Exactive instruments.
The coalescence artifact was completely removed by lowering the maximum
ion target for MS2 spectra from 1e6 to 2e5 without any losses in identification
depth or quantification quality of proteins
Measuring and Managing Ratio Compression for Accurate iTRAQ/TMT Quantification
Isobaric
mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive
multiplexed peptide/protein quantification technique in mass spectrometry.
However, accurate quantification of complex proteomic samples is impaired
by cofragmentation of peptides, leading to systematic underestimation
of quantitative ratios. Label-free quantification strategies do not
suffer from such an accuracy bias but cannot be multiplexed and are
less precise. Here, we compared protein quantification results obtained
with these methods for a chemoproteomic competition binding experiment
and evaluated the utility of measures of spectrum purity in survey
spectra for estimating the impact of cofragmentation on measured TMT-ratios.
While applying stringent interference filters enables substantially
more accurate TMT quantification, this came at the expense of 30%ā60%
fewer proteins quantified. We devised an algorithm that corrects experimental
TMT ratios on the basis of determined peptide interference levels.
The quantification accuracy achieved with this correction was comparable
to that obtained with stringent spectrum filters but limited the loss
in coverage to <10%. The generic applicability of the fold change
correction algorithm was further demonstrated by spiking of chemoproteomics
samples into excess amounts of <i>E. coli</i> tryptic digests
Chemical Proteomic Analysis Reveals the Drugability of the Kinome of <i>Trypanosoma brucei</i>
The protozoan parasite <i>Trypanosoma brucei</i> is the
causative agent of African sleeping sickness, and there is an urgent
unmet need for improved treatments. Parasite protein kinases are attractive
drug targets, provided that the host and parasite kinomes are sufficiently
divergent to allow specific inhibition to be achieved. Current drug
discovery efforts are hampered by the fact that comprehensive assay
panels for parasite targets have not yet been developed. Here, we
employ a kinase-focused chemoproteomics strategy that enables the
simultaneous profiling of kinase inhibitor potencies against more
than 50 endogenously expressed <i>T. brucei</i> kinases
in parasite cell extracts. The data reveal that <i>T. brucei</i> kinases are sensitive to typical kinase inhibitors with nanomolar
potency and demonstrate the potential for the development of species-specific
inhibitors
Interrogating the Druggability of the 2āOxoglutarate-Dependent Dioxygenase Target Class by Chemical Proteomics
The 2-oxoglutarate-dependent dioxygenase
target class comprises
around 60 enzymes including several subfamilies with relevance to
human disease, such as the prolyl hydroxylases and the Jumonji-type
lysine demethylases. Current drug discovery approaches are largely
based on small molecule inhibitors targeting the iron/2-oxoglutarate
cofactor binding site. We have devised a chemoproteomics approach
based on a combination of unselective active-site ligands tethered
to beads, enabling affinity capturing of around 40 different dioxygenase
enzymes from human cells. Mass-spectrometry-based quantification of
bead-bound enzymes using a free-ligand competition-binding format
enabled the comprehensive determination of affinities for the cosubstrate
2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate.
We also profiled a set of representative drug-like inhibitor compounds.
The results indicate that intracellular competition by endogenous
cofactors and high active site similarity present substantial challenges
for drug discovery for this target class