Journal of the American Chemical Society
Article
quantification is an accurate, reliable methodology for the
determination of protein abundance involving complex in vivo
samples.
AUTHOR INFORMATION
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Corresponding Author
CONCLUSION
ACKNOWLEDGMENTS
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A novel isobaric tag is developed for protein quantification,
referred to as CIT, with excellent demonstrated performance in
a range of typical proteomics investigations employing model
systems. The design of the CIT reagents is based on a novel
gas-phase fragmentation pathway reported here for the first
time. In this pathway, a nucleophilic displacement of the N3 of
the 1,2,3-triazole ring releases a stable reporter ion resulting
from formation of a six-membered ring. It should be noted that
DFT-calculated reaction energetics of reporter ion formation
are similar to those of backbone fragmentations in collisional
activation, permitting the effective quantification and sequenc-
ing simultaneously.
This work was supported by the National Science Foundation
(NSF) through grant CHE-0416381 (J.L.B.), the Beckman
Institute at California Institute of Technology (J.L.B., M.J.S.,
S.H., and R.J.L.G.), and the National Institutes of Health
(NIH) through grant RR 20004 (J.A.L.), and the Betty and
Gordon Moore Foundation (S.H. and R.J.L.G). Computational
resources for DFT results were kindly provided by the
Materials and Process Simulation Center at California Institute
of Technology. C.H.S. acknowledges a fellowship from the
Kwanjeong Educational Foundation. J.E.L. was supported by
the Ruth L. Kirschstein NRSA fellowship from the NIH
(CA138126).
In the preparation of CIT reagents, the mass of the reporter
ion can be easily tuned by varying azide groups in the
preparation of the 1,2,3-triazole ring via copper(I)-catalyzed
azide−alkyne cycloaddition (CuAAC), better known as click
reaction. The number of the possible isobaric tags is
determined by the number of isotope-tagged azide groups.
These azides can be prepared from halogenated alkyl groups,
which are also used for the alkylation of the linker amino acids,
reducing both the cost of reagents and the effort required for
the synthesis of isobaric tags. This modular feature expands the
possible number of combinations of CIT reagents. The
properties of CIT reagents can be tuned by using larger
isotope-coded halogenated alkyls that yield higher m/z reporter
ions, and these avoid the low mass cutoff problems normally
associated with ion trap mass spectrometers. Mixtures of light
and heavy CIT-labeled model peptides showed good linear
correlations with a two-orders-of-magnitude dynamic range.
Observed ratios of the light and heavy CIT-labeled protein
digests from the mixture of bovine serum albumin, ovalbumin,
α- and β-caseins, and lysozyme also exhibited good agreement
with the initial mixing ratio. Lastly, we have demonstrated the
applicability of CIT reagents in quantifying complex biological
samples using affinity-purified Cul1 ubiquitin ligase complexes
from HEK 293 cells.
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ASSOCIATED CONTENT
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S
* Supporting Information
Complete list of authors for ref 23; details of the synthesis and
characterization of compounds by ESI-MS and NMR; peptide
labeling of CIT reagents; Cul1 pull-down from HEK 293 cells;
mass spectrometry data processing; instrument conditions for
MALDI TOF, ESI-LTQ ion trap mass spectrometers for direct
infusion, and nanoLC-LTQ-Orbitrap experiments; details of
the computational methods; and tables of calculated Cartesian
coordinates and energies of model systems. This material is
(25) Treumann, A.; Thiede, B. Expert Rev. Proteomics 2010, 7, 647.
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dx.doi.org/10.1021/ja2099003 | J. Am. Chem.Soc. 2012, 134, 2672−2680