Revival of deuterium-labeled reagents for protein quantitation

Article abstract of DOI:10.1039/b906335h

A group of deuterium-labeled molecules that can be used for MS-based protein quantitation are synthesized, providing a cost-effective replacement of more expensive ¹³C- and ¹⁵N-labeled reagents.

Full text of DOI:10.1039/b906335h

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Revival of deuterium-labeled reagents for protein quantitationwz
Dexing Zeng and Shuwei Li*
Received (in College Park, MD, USA) 31st March 2009, Accepted 29th April 2009
First published as an Advance Article on the web 19th May 2009
DOI: 10.1039/b906335h
A group of deuterium-labeled molecules that can be used for
MS-based protein quantitation are synthesized, providing a cost-
effective replacement of more expensive 13C- and 15N-labeled
reagents.
analysis. During MS analysis, identical peptides differentially
labeled with DiART reagents are indistinguishable from each
other, thereby exhibiting a single peak. Once these precursor
ions are fragmented in tandem MS, the cleavable linker in the
tags is easily broken apart to produce a series of strong
reporter ions ranging from 114 to 119, allowing protein
quantitation by comparing the intensities of the six reporter
ions in the MS/MS spectra. Each DiART reagent also
contains the same number of 2H atoms (four per molecule)
that are placed next to hydrophilic groups, in order to
eliminate 2H-related chromatographic isotope effects. By using
2H as a coding isotope, the synthesis of DiART is greatly
simplified compared to other 13C- and 15N-labeled tags.
The synthesis of DiART reagents began with the prepara-
tion of b-alanine benzyl ester 1 (Scheme 1), which was then
coupled with Boc–leucine–NHS to yield compound 2. After
the Boc group was removed from compound 2, the primary
amine was treated under standard reductive methylation
conditions with formaldehyde and sodium cyanoborohydride,
followed by the removal of the benzyl protecting group by
hydrogenation to produce compound 3. The newly exposed
carboxylate group in compound 3 was activated by reacting
with NHS and DCC to yield the final product, compound 4.
Because all of these reactions have good yields, we were able to
synthesize DiART reagents within six steps in an overall yield
of 30%–40%, in contrast to 14-step and less than 1% yield
of TMT reagents.7 Furthermore, all of the isotope-labeled
starting materials can be purchased at low cost, making this
synthetic route a very cost-effective approach to prepare
DiART reagents.
Stable isotope labeling methods, such as ICAT, iTRAQ, and
SILAC, are widely used for the quantitative comparison of
proteins, providing versatile tools for proteomics research and
biomarker discovery.1 All of these approaches use reagents
2
that are coded with common isotope pairs, including H/1H,
13C/12C, and 15N/14N, to label identical peptides or proteins to
make them distinguishable by MS.2 However, 2H-labeled
molecules have recently been phased out and replaced by
13C- or 15N-coded tags because they can cause chromato-
graphic shift in reverse phase HPLC and compromise the
accuracy of quantitation by LC-MS/MS.3 Nevertheless, as
2H-labeled compounds are usually easier and less expensive
to synthesize than their 13C- or 15N-coded counterparts, it is
still of great interest to develop 2H-based technologies for
protein quantitation, provided 2H-related chromatographic
shift could be eliminated. For instance, a study to identify
structural features of 2H-containing molecules that are
2
responsible for their isotope effects indicates that placing H
atoms next to hydrophilic groups and minimizing the number
of 2H atoms in a molecule can reduce their contribution
to isotope effects, providing useful insight into the design of
2H-based tags that are irresolvable by HPLC.4
Here we report the development of a new type of 2H-labeled
reagent, named DiART, which can be used to quantitate
up to six protein samples concurrently (Fig. 1). Similar to
commercially available iTRAQ5 and TMT6 isobaric tags,
DiART reagents are a set of six structurally identical
molecules consisting of a reporter, a balancer, and a protein
reactive group. The mass of the reporter in a set is in the range
of 114–119, while the balancer in each molecule is changed
accordingly to offset the mass difference in the reporter to keep
the total mass of the reporter and the balancer in all reagents
the same. These reagents can covalently attach to the free
amine group of tryptic peptides and label them for MS/MS
Center for Advanced Research in Biotechnology, University of
Maryland Biotechnology Institute, Rockville, MD 20850, USA.
E-mail: liw@umbi.umd.edu
w Electronic supplementary information (ESI) available: Experiment
procedures. See DOI: 10.1039/b906335h
z Abbreviations: ICAT, isotope coded affinity tag; iTRAQ, isobaric
tag for relative and absolute quantitation; SILAC, stable isotope
labeling with amino acids in cell culture; DiART, deuterium isobaric
amine reactive tag; TMT, tandem mass tag; MALDI, matrix-assisted
laser desorption ionization; ESI, electrospray ionization; TFA,
trifluoroacetic acid; NHS, N-hydroxysuccinimide; Boc, tert-butoxy-
carbonyl; TEA, triethylamine; DCC, N,N0-dicyclohexylcarbodiimide.
Fig. 1 Structure of DiART tags consisting of a reporter, a balancer,
and a protein reactive group. Those positions containing heavy isotope
atoms (15N, 13C, 2H = D) in each reagent are underlined. When these
reagents are fragmented in MS/MS, they generate strong reporter ions
ranging from 114 to 119.
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Scheme 1 Synthesis of DiART reagents.
2
To demonstrate that the DiART reagents can eliminate H
isotope effects in reverse phase HPLC, we labeled phenyl-
alanine with each of the DiART reagents, mixed them
together, and injected them into a HPLC (Fig. 2). We obtained
a
broad peak and collected three time fractions for
MALDI-MS/MS analysis. All fractions generated the reporter
ions with identical relative intensities, implying that DiART-
labeled phenylalanines co-eluted and were irresolvable by
HPLC. Because phenylalanine, as a small single-residue
peptide, should be more prone to 2H-related isotope effects
than larger peptides, this result confirmed that DiART-labeled
tryptic peptides would not display any chromatographic shift.
To make DiART reagents useful for proteomics applica-
tions, they must be compatible with standard MS/MS data
analysis programs used for protein identification, such as
Mascot8 and SEQUEST.9 We decided to test DiART reagents
with Mascot because this database search engine is widely used
in the proteomics community and its latest release (version 2.2)
includes new features for protein quantitation that can be
easily modified to fit different needs. Therefore, we prepared
a three-protein mixture, including bovine serum albumin,
bovine catalase, and chicken ovalbumin. These proteins were
digested with trypsin after their cysteine residues were reduced
and alkylated under denaturing conditions. The resulting
peptides were labeled with six DiART reagents, respectively,
and then mixed at a 1 : 1 : 1 : 1 : 1 : 1 ratio. The labeled peptides
were then fractionated with a capillary HPLC and analyzed
with MALDI-MS/MS (Fig. 3a and b). The peak lists containing
their m/z values and intensities extracted from MS data
Fig. 3 (a) Capillary HPLC chromatogram of the tryptic peptide
mixture after DiART-labeling. A220
= absorbance at 220 nm.
(b) MS/MS spectrum of one peptide chosen from chicken ovalbumin
(Ova). The asterisk on the N-terminus of the peptide sequence
(GGLEPINFQTAADQAR) indicates that it is labeled with DiART
tags. This peptide was identified by Mascot with a high-confidence
score. Those b and y fragments are indicated. The inset is the expanded
spectrum in the range of 113–121. Relative ratios of six reporter ions
were also obtained from Mascot. Ir = relative intensity. m/z = mass-
to-charge ratio. (c) Relative ratios (115 : 114 circle, 116 : 114 star,
117 : 114 diamond, 118 : 114 square, 119 : 114 plus) of reporter ions
from five other peptides whose sequences and origins are shown.
BSA = bovine serum albumin. Cat = bovine catalase.
peptide labeled with different DiART reagents was obtained at
a 1.00 : 1.03 : 0.99 : 0.96 : 0.88 : 1.20 ratio after the isotope
impurity of DiART reagents was corrected, showing only a
small deviation from the known 1 : 1 : 1 : 1 : 1 : 1 ratio.10 After
five other peptides were quantified similarly to obtain enough
data that are statistically meaningful, the average coefficient of
variation was calculated as 0.11 (Fig. 3c), which was compar-
able to TMT and iTRAQ tags.11 In addition, we observed that
the cleavable linker in the DiART reagents was exceptionally
easy to fragment and the reporter ions were usually pre-
dominating peaks in most MS/MS spectra, greatly contributing
to the accuracy of quantitation because these signature ion
peaks have high signal-to-noise ratios.12
were sent to
a custom-configured Mascot server to
obtain both the identity and the quantity of peptides simulta-
neously. For example, a peptide from chicken ovalbumin
(GGLEPINFQTAADQAR) was identified with high
confidence. At the same time, the relative abundance of this
In summary, DiART reagents confirm that 2H-associated
chromatographic isotope effects can be eliminated if 2H-containing
molecules are properly designed. The protocol for protein identi-
fication and quantitation based on these reagents can be easily
incorporated into Mascot, one of the most widely used software
packages for MS/MS data analysis. Therefore, DiART reagents
offer a cost-effective approach for protein quantitation. Currently,
we are testing the performance of DiART-labeled peptides in
other types of mass spectrometers, such as ESI-based LTQ-
Orbitrap, and will report our results in due course.
Fig. 2 (a) HPLC chromatogram of a mixture of phenylalanines that
are differentially labeled with six DiART tags. Three time fractions
(1–3) were collected for MALDI-MS/MS analysis. A220 = absorbance
at 220 nm. (b) MS/MS spectra of three fractions. Each spectrum has
six reporter ion peaks (114.14, 115.14, 116.15, 117.15, 118.17, 119.17).
Ir = relative intensity. m/z = mass-to-charge ratio.
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