Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification

Article abstract of DOI:10.1021/ja2099003

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b-and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d₅-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Full text of DOI:10.1021/ja2099003

Article
pubs.acs.org/JACS
Click Chemistry Facilitates Formation of Reporter Ions and Simplified
Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein
Quantification
Chang Ho Sohn,† J. Eugene Lee,‡ Michael J. Sweredoski,§ Robert L.J. Graham,§ Geoffrey T. Smith,§
Sonja Hess,§ Gregg Czerwieniec,∥ Joseph A. Loo,⊥,# Raymond J. Deshaies,‡ and J. L. Beauchamp*,†
‡
†Division of Chemistry and Chemical Engineering, Division of Biology, and §Proteome Exploration Laboratory, Beckman Institute,
California Institute of Technology, Pasadena, California 91125, United States
⊥
#
∥Molecular Instrumentation Center, Department of Chemistry and Biochemistry, and Department of Biological Chemistry,
David Geffen School of Medicine, University of California at Los Angeles (UCLA), Los Angeles, California 90095, United States
S
* Supporting Information
ABSTRACT: We report the development of novel reagents
for cell-level protein quantification, referred to as Caltech
isobaric tags (CITs), which offer several advantages in
comparison with other isobaric tags (e.g., iTRAQ and
TMT). Click chemistry, copper(I)-catalyzed azide−alkyne cyclo-
addition (CuAAC), is applied to generate a gas-phase cleavable
linker suitable for the formation of reporter ions. Upon
collisional activation, the 1,2,3-triazole ring constructed by
CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter
ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined
by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the
nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino
acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are
similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the
development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded
precursor, allyl-d5-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of
the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via
N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of
isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping
with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The
applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.
Yet, current label-free quantification approaches require highly
consistent analyses, which are mostly hampered by fluctuations
in ionization efficiencies and difficulties in processing discrete
MS data.8,9
INTRODUCTION
■
Recent achievements in mass spectrometry (MS)-based
proteomics have provided essential methodologies for a deeper
understanding of protein expressions in cells.1,2 MS-based
proteomics allows high-throughput identification and quantifi-
cation of proteins of interest. Currently, state-of-the-art liquid
chromatography (LC)-MS instruments can analyze the whole-
cell yeast lysate within a day, identifying several thousand
distinct proteins.3,4 Quantitative approaches in MS-based
proteomics aim to investigate the relative and absolute
expression levels of proteins in cells.5,6 By employing those
approaches, various biological processes can be monitored by
tracking changes in protein expression.7
The simplest method for quantitative mass spectrometric
measurement is the label-free analysis. After successive runs of
samples of interest under the same instrument conditions,
protein abundances are determined by either integrating ion
chromatograms or spectral counting of peptide MS signals.8
In another label-free approach (which is also often performed
with internal standards and is thus not label-free), selected
reaction monitoring (SRM), and its extension plural multiple
reaction monitoring (MRM),10 examines the transitions (i.e.,
one or more targeted fragment ions from the precursor ions)
by scanning specific mass regions using triple quadruple mass
spectrometers. These permit highly sensitive identification
and concomitant quantification of peptides.11−13 SRM,
however, requires preknowledge of fragmentation behaviors
of analytes, for which the dissociation pathways must be deter-
mined through tedious assays prior to actual SRM analyses.
Received: October 20, 2011
Published: January 5, 2012
© 2012 American Chemical Society
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In addition, the high cost for the preparation of required
synthetic peptides may limit its wide application in shotgun
proteomics involving complex mixtures. SRM also suffers from
problems associated with fluctuations in ionization efficiencies
and chromatographic reproducibility.
MS/MS analyses. Preparing each part of the tag with various
isotopomers, in principle, allows for the facile construction of
multiplexed reagents capable of quantifying multiple samples in
a single MS analysis. The primary amines that are usually the
target functional groups for isobaric tag labeling exist in
virtually all peptides (i.e., the N-terminal amine and ε-amine of
lysine side chain), enabling researchers to investigate the whole
proteome. Isobaric tags have been employed for quantification
of tissue samples as well as samples from human, where
metabolic labeling methodology is not an option.26
In spite of the improvements achieved by isobaric tags, their
applications have been somewhat constrained by the cost of
commercially available reagents.27 In addition, the number of
the current multiplexed isobaric tags (e.g., iTRAQ) is limited to
a maximum of eight24 due to their inherent designs and
difficulties in the synthesis of various isotope-coded functional
groups. The low mass cutoff in resonance type ion trap mass
spectrometers, one of the most popular proteomics platforms,
also hinders the simultaneous monitoring of reporter ions and
peptide sequence ions.
To address problems associated with label-free quantifica-
tion, stable isotopes are incorporated into samples to be used as
internal (or mutual) standards.14 A conceptual breakthrough
for protein quantification was achieved by Aebersold and co-
workers by introduction of isotope-coded tags that can be used
to selectively label peptide digests.15 In this approach, cysteine-
containing peptides from different sources are tagged by light
or heavy isotope-coded affinity tag (ICAT) reagents and
enriched from the complex mixture using an attached biotin
affinity tag. Because these tagged peptides share the same
physicochemical properties, they are not differentiated by
ionization and chromatography steps. Therefore, a simple
comparison of MS signal intensities between light and heavy
isotope-coded peptides directly yields the relative protein
expression levels.
The design of new isobaric tags is hindered by the fact that
very few low-energy fragmentation pathways suitable for pro-
duction of the reporter ions are known in peptide MS/MS.
One of the widely known fragmentation pathways, the pre-
ferential cleavage of the aspartic acid−proline peptide bond
via a salt-bridged intermediate28,29 was applied in the first
TMT report (Scheme 1, I).21 In iTRAQ, the formation of the
Another quantification approach takes advantage of in vivo
metabolic incorporation of isotope labels.16−18 Among various
metabolic labeling methods, stable isotope labeling with amino
acids in cell culture (SILAC)19 has been popular due to its
simplicity in the incorporation of stable isotopes for
mammalian cells. Two cell populations are grown with identical
culture media except for stable isotope-labeled amino acids
(e.g., 13C- and/or 15N-labeled lysine and/or arginine). The
resulting heavy isotope-coded cell populations behave identi-
cally with their light isotope-coded controls. After applying a
perturbation to one of the light or heavy cell populations, the
two samples are combined for MS analysis. Direct comparison
of the peptide signals from the light and heavy labeled cell
populations yields the relative protein expression levels. Unlike
ICAT, which quantifies only cysteine-containing peptides,
metabolic labeling is capable of quantifying the global
proteome, because all tryptic peptides are labeled with
isotope-coded lysine and/or arginine.20
Scheme 1
The LC-MS ion signals from isotope-coded peptides in both
ICAT and metabolic labeling methodologies are divided into
two signals for each labeled peptide, causing an increase in the
complexity of MS scans and a reduction in the sensitivity of
subsequent sequence analysis by tandem mass spectrometry
(MS/MS). As an alternative chemical-labeling method, isobaric
tags, such as tandem mass tag (TMT)21,22 and isobaric tags for
relative and absolute quantification (iTRAQ),23,24 were
developed. An isobaric tag is composed of three parts: the
reporter ion group, the mass balance group, and the reactive
group.25 A clever feature in the design of these approaches is
that the combined mass of the reporter ion group and the mass
balance group is isobaric despite each part having different
masses. When the identical peptides from different biological
experiments are labeled by isobaric tags, they possess the same
masses, appearing as a single peak. Mass differentiated reporter
ions are produced and detected in MS/MS scans, in which their
relative intensities reflect the initial amounts of each peptide
from the original sources. All backbone fragment ions are also
isobaric, allowing simultaneous peptide sequencing and
quantification. One advantage of isobaric tags over ICAT-
and metabolic labeling-based methodologies, especially when
quantifying more than two system states, is that the combined
signals from multiple biological samples reduce the complexity
of the MS scans and increase the sensitivity for the following
reporter ions proceeds through the facile N-methylpiperazine-
acetyl bond-mediated cleavage process (Scheme 1, II). Most
isobaric tags that have been proposed to provide cheaper
synthetic routes are still based on similar tertiary amine-
branched methylene−amide bonds.30−32 Designing suitable
gas-phase fragmentation pathways is thus a critical task to
facilitate the development of novel isobaric tags and other mass
spectrometric analyses. Yet, to the best of our knowledge, it
has not been reported that one can intentionally design small
molecular tags using bio-orthogonal chemistry whose pep-
tide conjugates lead to highly selective gas-phase reactions,
generating a reporter ion.
Here, we report a novel isobaric reagent (Scheme 2), referred
to as Caltech isobaric tag (CIT), that is based on a newly found
gas-phase cleavable linker constructed by copper(I)-catalyzed
azide−alkyne cycloaddition (CuAAC) or simply click chem-
istry,33−35 affording a significant advance in cost-effective MS-
based protein quantification methodology.
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spectrometry. The design of a duplex CIT reagent is described,
and the applicability of the reagent is validated in model
systems using various mass spectrometers.
Scheme 2
METHODS
■
The synthetic route for generating CIT reagents is depicted in Figure 1.
Briefly, the isotope-coded isobaric tags were prepared via sequential
N-alkylations of L-alanine methyl ester by 6-hexynyl iodide and allyl-d0
or d5 bromides, CuAAC of mass balanced allyl-d0 or d5 azides, and
deprotection/activation of methyl ester to N-hydroxysuccinimidyl
ester. The resulting CIT reagents were conjugated to peptides and
analyzed by various mass spectrometers. The detailed synthetic
scheme and experimental methodologies can be found in Supporting
Information.
RESULTS AND DISCUSSION
■
CIT has several advantages over current isobaric labeling
reagents. Most importantly, CIT is easy to synthesize, and the
possible number of multiplexed isobaric tags, in theory, is
unlimited. Energetics for the reporter ion and peptide sequence
ion formations are balanced, guaranteeing simultaneous
quantification and sequencing of target peptides. CIT is
inspired by the observation of a highly selective gas-phase
fragmentation triggered by a nucleophilic displacement of the
N3 nitrogen in the 1,2,3-triazole ring (Scheme 3) produced by
Rationale of CIT Design. At the inception of this study, a
key goal was to find an appropriate gas phase fragmentation
pathway for the formation of the reporter ions. At that time, we
were interested in the application of bio-orthogonal CuAAC
reactions to MS-based proteomics studies. Observation of a
highly selective gas-phase fragmentation triggered by a
nucleophilic displacement of the N3 nitrogen in the 1,2,3-
triazole ring competitive with the formation of b- and y-type
ions in collision-induced dissociation (CID) of covalently
labeled peptides inspired us to create novel isobaric tags
(Scheme 3). In multiply protonated CIT-labeled peptides, the
tertiary amine in the CIT reagent would be protonated due to
its higher proton affinity than most backbone amides and
amino acid side chains. A nucleophilic displacement of the N3
of the 1,2,3-triazole ring at the α-carbon position of the
protonated N,N-alkylated alanine residue in the CIT reagent
releases a stable quaternary ammonium reporter ion, forming a
six-membered ring.
Scheme 3
The energetics of reporter ion formation is investigated by
density functional theory calculations (Figure 2). If this process
is significantly favored compared to backbone fragmentation,
then less sequence information would be acquired by having
fewer and weaker intensity b- and y-type ions in the MS/MS
spectrum. It is desirable that activation parameters associated
with reporter ion formation are balanced with those of
backbone fragmentation. This ensures that accurate protein
quantification is achieved while not reducing sequencing
CuAAC. Previously, the 1,2,3-triazole ring is known to be inert
in the peptide gas-phase fragmentation.36−38 We show that by
designing proper linkers, it is possible to induce highly selective
gas-phase reactions to be useful in peptide tandem mass
Figure 1. Synthesis of CIT reagents. (a) THF, K2CO3, TEAI, reflux 18 h, 56%. (b) THF, K2CO3, TEABr, reflux 18 h, R1 = Allyl-d0-bromide (4a),
56%, R1 = Ally-d5-bromide (4b), 67%. (c) 0.4 equiv Na ascorbate, 0.1 equiv CuSO4, 0.01 equiv TBTA, DMSO/H2O, RT 4 h, R2 = Allyl-d5-azide
(5a), 72% (heavy tag), R2 = Allyl-d0-azide (5b), 69% (light tag). (d) 2 M KOH, THF, RT overnight, quantitative (heavy tag, 6a), 97% (light tag, 6b).
(e) TFA-NHS, DMF, RT overnight, 24% (heavy tag, 7a), 23% (light tag, 7b). THF = tetrahydrofuran, TEAI = tetraethylammonium iodide, TEABr
= tetraethylammonium bromide, TBTA = tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, DMSO = dimethyl sulfoxide, TFA-NHS =
trifluoroacetic N-hydroxysuccinimide ester, DMF = N,N-dimethyl formamide.
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Figure 2. Energetics of reporter ion formation. The model system,
N,N-dimethyl-4-(1-methyl-1H-1,2,3-triazol-4-yl)butan-1-amine was
chosen for calculation. Geometry optimization and thermochemical
calculations were performed at the B3LYP/6-311++G(d,p) level of
theory. The shaded area indicates the range of enthalpies of activation
for amide cleavage of protonated peptides to form b- and y-type ions
via collisional activation.
efficacy using MS/MS. In our calculation model, the formation
of the reporter ion is simulated by the N-protonated N,N-
dimethyl-4-(1-methyl-1H-1,2,3-triazol-4-yl)butan-1-amine. At
the B3LYP/6-311++G(d,p) level of theory, the reaction barrier
and enthalpy at 1 atm and 298.15 K are determined as 33.7 and
13.3 kcal/mol, respectively (Figure 2). The usual reaction
barrier for amide bond cleavage in protonated peptides ranges
from 25 to 40 kcal/mol.39 Therefore, it is anticipated that most
backbone cleavages will occur competitively with formation of
the reporter ion. In addition to the formation of reporter ions,
this feature also validates the usefulness of the observed
fragmentation pathway for other applications involving mass
spectrometry, such as the controlled release of affinity tags,
used in the separation of targeted peptides, by collisional
activation.
Deriving advantage from the described gas-phase cleavage
reaction to form reporter ions, we proceeded to design novel
isobaric tags. Figure 3 depicts the structure of CIT, the
construction of the theoretical N-plex reagents, and the
schematic drawing of their application to peptide conjugates.
CIT is composed of three parts: the reporter ion group, the
mass balance group, and the amine reactive group found in
other commercially available isobaric tags.
The major improvement of CIT that distinguishes it from
other isobaric tags is the modularization of the isotope-coded
residues, both for the mass balance and the reporter ion groups.
Any functional groups (R) that do not contain other reactive or
interfering functionalities can be implemented into the mass
balance group by inserting a good leaving group, such as
bromine, iodine, or tosylate. Via a simple SN2 reaction in the
mild conditions employing DMF/NaN3, the isotope-coded
reporter ion group can be easily prepared from an activated R
group that is used for the mass balance group (Figure 3). Each
isobaric pair of Rm−X (X: leaving group) and Rn−N3 forms a
building block for an isobaric tag with a certain reporter ion
mass (Rn + 123 Da). By preparing a set of the N different
Figure 3. Design and structure of CIT reagents. (a) The components
of N-plex CIT reagents: the reporter ion group, the mass balance
group, and the amine reactive group. (b) Each reporter ion and mass
balance group can be prepared from a series of isotope-coded
iodinated Rn groups. CIT-labeled peptides are fragmented by various
ion activation methods (e.g., PQD and HCD), yielding the reporter
ions whose masses are Rn + 123 Da. (c) The duplex embodiment of
the CIT reagents in this report by using allyl bromide-d0 and d5. Note
that the reporter ion is formed regardless of the structure of the attached
Rn or Rm groups. (d) Schematic drawing for the quantification and
sequencing of 1:2 light and heavy CIT-labeled peptides by MS/MS.
isotope-coded R−X, it is possible to construct the N-plex
isobaric reagents. This modularity of CIT significantly reduces
the effort and cost of synthesis. In addition, the mass of the
reporter ion is tunable; this property enables us to bypass the
mass cutoff problem in ion trap mass spectrometers and target
open windows of m/z values normally found in peptide MS/MS
(e.g., sequence and immonium ions or internal fragments).
This general experimental and synthetic methodology is
applied to the creation of a prototype duplex CIT reagent using
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allyl bromide-d0 and d5 as the isotope-coded starting materials.
Duplex CIT reagents were synthesized, with stable isotopes
having the reporter ions at m/z 164 and 169, respectively. We
adopt the NHS group for facile amine-reactive coupling to
peptides as in other commercially available isobaric tags. NHS
has been popular in bioconjugation due to the compatibility
with most biological buffer solutions. Most importantly, its
target functional groups (N-termini of peptides and the
ε-amine of lysine) are ubiquitous among tryptic peptides.
The size of the overall modification by this duplex CIT reagent
is 279 Da, which is not much larger than most of the
commercially available isobaric tags (iTRAQ 4-plex, 144 Da;
TMT 6-plex, 224 Da; iTRAQ 8-plex, 304 Da).
MS/MS of CIT-Labeled Peptides. The light and heavy
duplex CIT reagents were used to label the model tryptic
peptide, HSDAVFTDNYTR. Figure 4 depicts matrix-assisted
Figure 4. MALDI TOF MS spectra of (a) light and (b) heavy CIT
reagent labeling of the model peptide, VIP(1−12), HSDAVFTD-
NYTR. The labeling reaction was performed for 2 h and quenched
using 0.1 M hydroxylamine. The conversion yield was approximately
∼99%. Quenching by 0.1 M hydroxylamine reverses unwanted
byproduct, which contain CIT reagent conjugation on tyrosine
residues. Some of the impurities were observed, but their contributions
were appropriately considered for the calculation of H/L ratios.
Figure 5. PQD of doubly and triply protonated CIT- and iTRAQ-113-
labeled model peptide ions, HSDAVFTDNYTR with 1:1 = light:heavy
ratio. Both (a) and (b) show abundant reporter ions at m/z 164 and
169, respectively, whereas the intensities of the reporter ions at m/z
113 in (c) and (d) are relatively small, indicating the favored
energetics of the fragmentation pathway used for the CIT reporter
ions. PQD of the triply protonated peptide ions in (b) and (d) also
yields abundant sequence ions. For PQD of the doubly protonated
peptide ions, the sequestered protons at the CIT residue and the
arginine side chain increase the barrier for amide bond cleavage,
yielding less backbone fragments. Experimentally observed reporter
ion ratios (H/L) were 0.80 for 2+ and 0.81 for 3+, compared to the
expected ratio of 1.0. These deviations likely result from initial
experimental mixing errors (see the main text for the details).
laser desorption/ionization (MALDI) time-of-flight (TOF) MS
spectra of CIT-labeled model peptides. The masses of CIT-
labeled peptides are 279 Da larger than the original peptides as
expected (Figure 4). The labeling yields of light and heavy CIT
reagents are both ∼99% estimated by the peak height
comparison between unmodified and CIT-labeled peaks in
the MALDI TOF MS spectra (Figure 4). The exact masses of
light and heavy labeled peptides are identical, appearing as one
peak in all mass spectrometric analyses.
Pulsed-Q dissociation (PQD) in the linear ion trap of a
LTQ-Orbitrap classic mass spectrometer was used to fragment
protonated CIT-labeled peptide ions produced by electrospray
ionization (ESI). PQD of the 1:1 mixture of the heavy and light
CIT-labeled peptides in the ion trap generates abundant
reporter ions at m/z 164.1 and 169.1 as well as sequence ions,
confirming N-terminal labeling (Figure 5a and b). PQD of the
triply protonated precursor ion yields abundant backbone
fragment ions along with reporter ions, but less prominent
backbone fragments are observed for the doubly protonated
precursor ion. These results are presumably caused by
sequestering of mobile protons at the CIT and arginine
residues, increasing the reaction barrier for backbone cleavage.
The H/L ratio of the CIT reporter ion is determined to be 0.8
in both +2 and +3 charge states (Figures 5a and b), which we
attribute to an initial experimental mixing error. Note that for
the PQD experiment, light and heavy labeled peptides are
prepared and purified separately by C18 ziptips and later mixed
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for linear response testing with various mixing ratios to reduce
the effort for desalting each mixed sample. In the actual
quantification experiment, the sample mixing step is prior to
the C18 purification to avoid dissimilar sample loss. This
systematic error appears as the y-axis intercepts in the linear
fitting lines in Figure 6.
To address this possibility, the retention times in nanoLC were
measured by injection of the 1:1 mixture of the light and heavy
CIT-labeled model peptides. For monitoring higher energy
collisional dissociation (HCD) signals of the reporter ions, the
MS/MS analysis was performed repeatedly by turning off the
data-dependent acquisition mode. Figure 7 depicts nanoLC
For comparison of reporter ion yields to the CIT reagent, the
iTRAQ-113 reagent having no isotope substitution was
conjugated to the same model peptide, and the resulting
peptides were subject to PQD (Figure 5c and d). The overall
sequence coverage of the iTRAQ-labeled peptide by PQD is
very similar to that of the CIT-labeled peptide. Yet, the relative
reporter ion intensities in the PQD spectra of the doubly and
triply protonated iTRAQ-labeled peptide ions are lower than
those of the CIT-labeled peptides (Figure 5c and d). This result
indicates that the process for reporter ion formation of CIT is
slightly favored over that of iTRAQ, enabling more reliable
quantification with the CIT reporter ions.
Figure 7. The nanoLC chromatograms of (a) MS1 scan, (b) m/z 164
(red) and 169 (blue) reporter ions observed in HCD scans generated
by the 1:1 mixture of light and heavy CIT labeled model peptides,
HSDAVFTDNYTR, and (c) a close look of the reporter ion counts
and 169/164 ratios around 17.3 min. The base peaks in all
chromatograms are related to CIT-labeled model peptides. Note
that the red and blue lines are very similar, indicating almost identical
chromatographic behavior of light and heavy CIT-labeled peptides
Figure 6. The linear fitting trend lines obtained by calculating the log 2
of summations of peak heights at m/z 164 and 169 for y-axis and the
log 2 of intended initial mixing ratios for x-axis. Data points are
obtained from the PQD spectra of the precursor ions with the charge
states of (a) 2+ and (b) 3+ using a profile mode, and (c) 2+ and (d)
3+ using
a centroid mode. Relatively large (∼0.4−0.5)
y-axis intercepts in all figures are originated from systematic sources,
such as initial experimental mixing errors (see the main text for the
details). Therefore, the overall linearity (slopes = ∼1.0) and quality of
fitting (R2 = ∼0.99) are not affected.
(0.97
0.05). The peak at 14.9 min in (a) is a nonlabeled model
peptide. The peak appearing around 19.4 min in (a) is from CIT-
labeled peptide fragments, AVFTDNYTR. Because this fragment
peptide existed in each peptide stock independently, their initial
mixing ratios were different from the original intact peptide, yielding
dissimilar 169/164 ratios in (b). The small peak at 18.6 min is
associated with doubly CIT-labeled peptides including tyrosine and
N-terminal modifications.
Dynamic Range. To test the dynamic range of the CIT
reagent, we monitored reporter ion formation with various
ratios of light and heavy labeled peptides using PQD in a LTQ-
Orbitrap classic mass spectrometer. The PQD spectra of doubly
and triply charged CIT-labeled peptides were recorded in
profile and centroid modes, and the intensities of the reporter
ions were used to plot the linear dependency on the initial
mixing ratio (Figure 6, log 2−log 2 plot). The overall linearity
(slopes = ∼1.0) and quality of fitting (R2 = ∼0.99) indicate a
very good linear response of the CIT reagents from 1/9 to 9
H/L mixing ratios. This demonstrates roughly a two-orders-of-
magnitude dynamic range with the performance of the CIT
reagents for relative quantification.
chromatograms of the CIT-labeled peptides (Figure 7a) and
their HCD scans (Figure 7b and c) filtered by m/z 164 (red)
and 169 (blue) mass ranges to monitor the elution profiles of
the light and heavy CIT-labeled peptides. As seen in Figure 7b
and c, ion current profiles for both reporter ions appear
identical with no apparent differential tailing effect. The mean
H/L ratio of CIT-labeled model peptides eluted at 17 min is
determined as 0.97 0.05. This result indicates that both light
and heavy CIT-labeled peptides have the same chromatographic
properties, validating the suitability of the CIT reagents for
protein quantification using LC-MS platforms.
Chromatographic Separation. The tailing of deuterated
counterparts in LC elution profiles has been reported
previously and may affect the accuracy of quantification.30,40,41
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Protein Labeling. The applicability of the CIT reagent is
tested with model systems involving protein mixtures. Protein
digests prepared from the mixture of bovine serum albumin
(BSA), ovalbumin, α- and β-caseins, and lysozyme were used
for CIT labeling with the initial H/L mixing ratio of 1:1. The
nanoLC-LTQ-Orbitrap analyses using PQD (Orbitrap classic)
and HCD (Orbitrap velos) generally reproduce the initial
mixing ratio, in which hundreds of peptides tagged by CIT
reagents are quantified (Table 1). Importantly, both PQD and
Table 2. Quantification Results of CIT-Labeled CUL1
Complex
H/L
ratio
no. of quantified peptide
hits
confidence interval
(95%)
protein
ACTB
Cul1
1.02
0.94
1.00
1.00
0.89
0.87
1.07
1.06
0.92
0.78
0.80
1.59
1.02
0.96
0.50
0.62
13
11
11
11
9
(0.87, 1.20)
(0.62, 1.14)
(0.83, 1.18)
(0.61, 1.37)
(0.63, 1.20)
(0.60, 1.09)
(0.93, 1.17)
(1.02, 1.32)
N/A
POTEE
CAND1
ACTN4
ACTN1
FLNA
6
Table 1. Quantification Results of CIT-labeled Protein
Mixture
5
ACTA1
SPTAN1
UBB
3
2
no. of
quantified
1
N/A
H/L ratio
peptide hits
confidence interval (95%)
PQD HCD
CORO1C
SKP1
1
N/A
protein
PQD HCD PQD HCD
1
N/A
MYL6B
COPS8
COPS6
COPS2
1
N/A
BSA
0.95
0.87
1.05
0.89
1.06
0.95
0.82
1.04
0.91
1.06
45
44
4
119
48
27
25
17
(0.80, 1.19) (0.88, 1.03)
(0.73, 1.10) (0.65, 0.86)
(0.69, 1.19) (0.96, 1.26)
(0.69, 1.19) (0.70, 1.15)
(0.59, 1.93) (0.97, 1.29)
1
N/A
ovalbumin
lysozyme
α-S1-casein
β-casein
1
N/A
1
N/A
27
7
Lastly, the applicability of the CIT reagent for the
quantification of relative protein expression levels in vivo is
investigated. In this study, different amounts of Cul1 were
expressed in stable HEK 293 cells by treating two popula-
tions of cells with either 0.5 μg/mL of tetracycline for 1 h or
2.0 μg/mL for 4 h. These two samples were subject to Western
blot analysis. As shown in Figure 8, the level of Cul1 expression
HCD resulted in very similar heavy to light ratios, although the
confidence intervals of the medians at 95% confidence level
reported in PQD are relatively large (approximately 40% of the
observed medians). Judging from the same phenomenon
observed independently in the previous report using PQD for
the quantification of iTRAQ-labeled peptides,42 we believe that
the relatively large confidence intervals result, in large part,
from the poor performance of PQD. Compared to PQD, HCD
in a LTQ-Orbitrap velos mass spectrometer systematically
yielded smaller confidence intervals (approximately 20% of the
observed medians) with more peptide identification, showing
its superior performance in both detection and quantification of
CIT-labeled peptides.
Next, the CIT reagent is applied to quantify biologically
relevant samples. Cul1 is a ubiquitin ligase that forms a large
protein complex with dozens of known binding partners.43 This
protein complex was purified from HEK 293 cells and
quantified using CIT after tryptic digest. To facilitate
purification of the Cul1 complex, we constructed a stable cell
line that expresses tandem-tagged Cul1 upon tetracycline
treatment.44 Trypsin digests of Cul1 protein complexes affinity
purified from the HEK 293 cell line were split with the ratio of
1:1 for labeling with heavy or light CIT reagents. The labeled
samples were combined, and the resulting mixture was analyzed
by HCD/CID in a LTQ-Orbitrap XL mass spectrometer. Table 2
lists identified proteins with the H/L ratios determined by
Mascot. The calculated medians are close to 1 for identified
proteins with multiple quantified peptide hits, indicating that
CIT is suitable for quantification of complex biological samples.
In quantifying more complex mixtures, such as whole cell
lysates, other isobaric tags have often yielded less reliable
quantification results by coincidently isolating impurities for
MS/MS analyses due to the high sample complexity.45 Our
CIT reagents may also suffer from interferences by having
overlaps among precursor ions in complex mixtures, resulting in
poor quantification results. By applying the gas-phase
purification via proton-transfer reaction methodology devel-
oped by Coon and co-workers46 or the MS3 analysis by Gygi
and co-workers,47 one can avoid these interferences in complex
mixture analysis using our isobaric tags.
Figure 8. Western blot analysis of differentially expressed HTBH
(His6, tobacco etch virus protease site, in vivo biotinylation sequence,
and another His6) tagged Cul1 from HEK 293 cells. The amounts of
GAPDH and p27 were analyzed as reference proteins. Differential
induction was performed by adding 0.5 or 2.0 μg/mL of tetracycline to
the growth medium for 1 or 4 h, respectively (lanes 1 and 2). Lanes 3
and 4 are the replicates of lanes 1 and 2, respectively. The relative Cul1
expression level was 1:5.3 for Lanes 1 and 2 and 1:5.2 for lanes
3 and 4. HCD/CID of CIT-labeled Cul1 digests yielded median H/L
ratio of 5.6 using 12 peptides. The geometric standard deviation was
2.2, and the confidence interval with 95% confidence was (5.3, 8.9).
differs by ratios of 5.3 (lane 2/lane1) and 5.2 (lane 4/lane 3),
respectively. For MS analyses, Cul1 was purified from the two
differentially expressed samples and digested by Lys-C/trypsin.
After CIT labeling, the resulting peptides were analyzed by
HCD/CID in an LTQ-Orbitrap XL mass spectrometer.42,48
The median for the H/L ratio of 12 Cul1 tryptic peptides is 5.6,
which agrees well with the ratio determined by Western blot
experiments. These results demonstrate that the CIT-based
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dx.doi.org/10.1021/ja2099003 | J. Am. Chem.Soc. 2012, 134, 2672−2680

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
■
Corresponding Author
jlbchamp@caltech.edu.
CONCLUSION
ACKNOWLEDGMENTS
■
■
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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