Multiple isotopic labels for quantitative mass spectrometry

Article abstract of DOI:10.1021/ac801654h

Quantitative mass spectrometry is often performed using isotopicalty labeled samples. Although the 4-trimethylammoniumbutyryl (TMAB) labels have many advantages over other isotopic tags, only two forms have previously been synthesized (i.e., a heavy form containing nine deuteriums and a light form without deuterium). In the present report, two additional forms containing three and six deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents coelute from reversed-phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these four tags allow for multivariate analysis in a single liquid chromatography/mass spectrometry analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated. The synthetic scheme is described in simple terms so that a biochemist without specific training in organic chemistry can perform the synthesis. The interpretation of tandem mass spectrometry data for the TMAB-labeled peptides is also described in more detail. The additional TMAB isotopic reagents described here, together with the additional description of the synthesis and analysis, should allow these labels to be more widely used for proteomics and peptidomics analyses.

Full text of DOI:10.1021/ac801654h

Anal. Chem. 2008, 80, 9298–9309
Multiple Isotopic Labels for Quantitative Mass
Spectrometry
Cain Morano, Xin Zhang, and Lloyd D. Fricker*
Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461
Quantitative mass spectrometry is often performed using
isotopically labeled samples. Although the 4-trimethylam-
moniumbutyryl (TMAB) labels have many advantages over
other isotopic tags, only two forms have previously been
synthesized (i.e., a heavy form containing nine deuteriums
and a light form without deuterium). In the present report,
two additional forms containing three and six deuteriums
have been synthesized and tested. These additional iso-
topic tags perform identically to the previously reported
tags; peptides labeled with the new TMAB reagents
coelute from reversed-phase HPLC columns with peptides
labeled with the lighter and heavier TMAB reagents.
Altogether, these four tags allow for multivariate analysis
in a single liquid chromatography/mass spectrometry
analysis, with each isotopically tagged peptide differing
in mass by 3 Da per tag incorporated. The synthetic
scheme is described in simple terms so that a biochemist
without specific training in organic chemistry can perform
the synthesis. The interpretation of tandem mass spec-
trometry data for the TMAB-labeled peptides is also
described in more detail. The additional TMAB isotopic
reagents described here, together with the additional
description of the synthesis and analysis, should allow
these labels to be more widely used for proteomics and
peptidomics analyses.
characterized using radioimmunoassays or related techniques.10
Although these techniques provided the ability to compare the
immunoreactive levels of a peptide in a large number of different
samples, there were three major drawbacks. First, the antisera
often cross-reacted with longer or shorter forms of the peptide,
and/or cross-reacted with post-translationally modified peptides
(i.e., phosphorylated, acetylated, etc.). Thus, it was difficult to be
sure of the specific form being measured with the antibody-based
assay. Second, it was very time-consuming to raise antisera to a
peptide, characterize the antisera, and set up a selective immu-
noassay to detect the peptide. Third, it was only possible to study
known peptides. In contrast, mass spectrometry enables hundreds
of peptides to be detected in a single experiment, with knowledge
of the precise molecular form being measured, and can detect
unknowns as well as previously identified peptides.
Some previous peptidomics studies have obtained an estimate
of the relative levels of peptides in two or more samples by
comparing the relative intensity among different LC/MS runs of
the peptides present in a complex sample.11-14 Although it may
be possible to achieve some accuracy with this nonisotopic
method, it is difficult to quantify small changes using this
technique. A more accurate method to measure small changes in
peptide levels involves stable isotopes.15-17 In a typical experiment
involving isotopes, peptides labeled with light isotopes (1H, 12C,
14N, and/or 16O) are combined with peptides labeled with heavy
isotopes (2H, 13C, 15N, and/or 18O) prior to MS and the relative
peak intensity of the two forms provides an accurate indication of
A large number of bioactive peptides are known to play
important physiological roles in a variety of organisms, ranging
from yeast to humans.1-4 Some peptides function in communica-
tion between cells (neuropeptides, peptide hormones, growth
factors, and mating factors), whereas others function as toxins to
capture prey or to repel predators (snake venoms, marine
organism toxins, and antimicrobial peptides).
Developments in mass spectrometry (MS) in the past decade
have led to a dramatic improvement in the detection of bioactive
peptides.3,5-9 Prior to mass spectrometry, peptides were primarily
(6) Baggerman, G.; Verleyen, P.; Clynen, E.; Huybrechts, J.; De Loof, A.;
Schoofs, L. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2004, 803,
3–16
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P. E. Biochem. Soc. Trans. 2007, 35, 588–593.
(8) Li, L.; Kelley, W. P.; Billimoria, C. P.; Christie, A. E.; Pulver, S. R.; Sweedler,
J. V.; Marder, E. J. Neurochem. 2003, 87, 642–656.
(9) Rubakhin, S. S.; Churchill, J. D.; Greenough, W. T.; Sweedler, J. V. Anal.
Chem. 2006, 78, 7267–7272.
(10) Chard, T. An Introduction to Radioimmunoassay and Related Techniques,
3rd ed.; Elsevier: Amsterdam, The Netherlands, 1987.
(11) Skold, K.; Svensson, M.; Norrman, M.; Sjogren, B.; Svenningsson, P.;
Andren, P. E. Proteomics 2007, 7, 4445–4456
(12) Husson, S. J.; Janssen, T.; Baggerman, G.; Bogert, B.; Kahn-Kirby, A. H.;
Ashrafi, K.; Schoofs, L. J. Neurochem. 2007, 102, 246–260
(13) Husson, S. J.; Clynen, E.; Baggerman, G.; Janssen, T.; Schoofs, L.
J. Neurochem. 2006, 98, 1999–2012
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Acad. Sci. U.S.A. 2001, 98, 9971–9976
(15) Julka, S.; Regnier, F. E. Briefings Funct. Genomics Proteomics 2005, 4, 158–
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(16) Fricker, L. D.; Lim, J.; Pan, H.; Che, F.-Y. Mass Spectrom. Rev. 2006, 25,
327–344
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* Corresponding author. E-mail: fricker@aecom.yu.edu. Phone: 718-430-
4225. Fax: 718-430-8922.
.
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9298 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008
10.1021/ac801654h CCC: $40.75 2008 American Chemical Society
Published on Web 11/05/2008

the relative levels of each peptide in the sample. For studies on
cultured cells, as well as some lower organisms, differential
isotopic labeling can be obtained by growing one sample in normal
media while the other gets growth media supplemented with
heavy isotopes.18,19 However, for studies on mammals, the heavy
isotopes are prohibitively expensive. Therefore, studies on mam-
malian peptides have generally used postextraction labeling of the
peptides with isotopic tags.15
A number of isotopic tags have been developed for the
quantification of proteins and peptides.15,20-26 The isotopically
coded affinity tag (ICAT) labels were developed for proteomics
studies; this reagent reacts with the thiol group of Cys and also
contains a biotin affinity tag to allow for purification of the labeled
peptide.20 However, because most peptides do not contain thiol
groups, the ICAT labels will not work for peptidomics analysis.
Instead, peptidomics studies have focused on isotopic tags that
label primary and secondary amines which are contained on the
N-terminus and on the side chain of internal Lys residues (unless
acetylated or otherwise modified).22 Commercially available
reagents that label amines and can be used for quantitative
peptidomics include acetic anhydride, succinic anhydride, and
iTRAQ reagents.15,20-22,22,27 However, these suffer from various
drawbacks. Although relatively inexpensive, the acetic and suc-
cinic anhydrides convert positively charged amines on a peptide
into neutral sites (acetyl) or negative charges (succinyl), and for
some peptides this results in a weak signal when analyzed by MS
in positive ion mode.22 Furthermore, peptides labeled with the
heavy forms of these anhydrides do not always coelute on LC/
MS with the light forms, and quantitation of these peptides is less
accurate than for those isotopically tagged peptides that do
coelute.22 The iTRAQ reagent solves these latter problems, but it
is very expensive, difficult to synthesize, and only shows signals
when peptides are subjected to collision-induced dissociation
(CID) and tandem mass spectrometry (MS/MS).21 Because many
peptides can be detected in MS spectra of complex mixtures, but
only one of these is analyzed by MS/MS at any given time, the
iTRAQ reagent will miss those peptides not selected for MS/MS
analysis.21
Figure 1. Synthetic scheme of TMAB labels. Step (a) is the alkylation
of γ-aminobutyric acid (GABA) using methyl iodide or one of its
deuterated isotopes. This generates a trimethylammonium iodide salt,
which is washed with chloroform and then converted to the chloride
salt by acidification with HCl. Step (b) is the activation of the acid by
formation of the N-hydroxysuccinimide (NHS) ester using dicyclo-
hexylcarbodiimide (DCC).
the label came in more than just two forms (i.e., “heavy” and
“light”) so that multiple comparisons could be performed within
the same LC/MS run.
The 4-trimethylammoniumbutyryl (TMAB) labels were devel-
oped by Regnier’s group and contain a quaternary amine labeled
with methyl groups which imparts a permanent positive charge
on the peptide.23 Although the TMAB labels are close to ideal,
there are several problems. First, the synthesis is fairly simple,
but the description of the synthesis in the literature is difficult
for a nonchemist to follow. Second, the isotopic tags often partially
decompose to stable intermediates upon CID and MS/MS
analysis, and this complicates computer and manual interpretation
of the data.22,28 Finally, only heavy and light forms of these TMAB
labels have been previously reported.23 In the present study, we
report the synthesis and evaluation of additional isotopic TMAB
tags that allow four samples to be compared in the same LC/MS
run. In addition, we describe the synthesis in more detail, with
instructions for a nonchemist and tips on how to get high yields.
Finally, we describe the problems with data interpretation,
providing guidelines for manual analysis. Although developed for
quantitative peptidomics applications, these labels should be useful
for proteomics studies too, much like the iTRAQ reagents but
with several advantages.
The ideal isotopic label has several key properties. The label
should react with amines efficiently and quantitively but also have
a long shelf life, be inexpensive and either commercially available
or easy to synthesize, and maintain the positive charge on the
labeled amine. In addition, the isotopically labeled peptides should
coelute on LC and be stable to MS. Finally, it would be ideal if
MATERIALS AND METHODS
Reagents. γ-Aminobutyric acid (GABA), potassium bicarbon-
ate, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide, meth-
yl iodide (sold as iodomethane) and its -d1, -d2, and -d3 isotopes,
dimethyl sulfoxide (DMSO), sodium hydroxide, glycine, and
hydroxylamine hydrochloride were purchased from Aldrich
Chemical Co. (Milwaukee, WI). Acetonitrile of HPLC grade,
anhydrous solvents such as tetrahydrofuran (THF), acetonitrile,
methanol, and acetone were obtained from Fisher Scientific (Fair
Lawn, NJ). Hydrochloric acid (6 N, sequanal grade, constant
boiling), trifluoroacetic acid, and formic acid were purchased from
Pierce (Rockford, IL). Water was purified with a Synergy UV
system (Millipore S.A.S., Molsheim, France).
(18) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.;
Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376–386
(19) Che, F.-Y.; Yuan, Q.; Kalinina, E.; Fricker, L. D. J. Neurochem. 2004, 90,
585–594
(20) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R.
Nat. Biotechnol. 1999, 17, 994–999
.
.
.
(21) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan,
S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz,
P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol.
Cell. Proteomics 2004, 3, 1154–1169
(22) Che, F.-Y.; Fricker, L. D. J. Mass Spectrom. 2005, 40, 238–249
(23) Zhang, R.; Sioma, C. S.; Thompson, R. A.; Xiong, L.; Regnier, F. E. Anal.
Chem. 2002, 74, 3662–3669
(24) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal. Chem. 2003, 75,
6843–6852
(25) Schmidt, A.; Kellermann, J.; Lottspeich, F. Proteomics 2005, 5, 4–15
(26) Mason, D. E.; Liebler, D. C. J. Proteome Res. 2003, 2, 265–272
(27) Che, F.-Y.; Fricker, L. D. Anal. Chem. 2002, 74, 3190–3198
.
.
Synthesis of TMAB Labels: Overview. The overall reaction
scheme is shown in Figure 1 and is described in detail below both
in a standard protocol for chemists as well as a less technical
.
.
.
.
(28) Che, F. Y.; Zhang, X.; Berezniuk, I.; Callaway, M.; Lim, J.; Fricker, L. D. J.
.
Proteome Res. 2007, 6, 4667–4676.
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008 9299

then dried under vacuum. Overall, the reaction produced 2.02 g
(11.1 mmol, 52% yield) of TMAB acid.
Biologist Protocol. (1) Prepare the reagents and equipment:
Place a 500 mL round-bottom flask, glass 10 mL pipet, excess
potassium bicarbonate, glass flask stopper, and Teflon stir bar in
a vacuum oven at approximately 50 °C overnight; after ap-
proximately 16 h, turn off heat and allow to cool to room
temperature under vacuum. Place excess GABA in an Erlenmeyer
flask inside a desiccator, then place it under high vacuum at room
temperature overnight. Methyl iodide and isotopes are liquids and
should remain sealed, refrigerated, and under inert gas if possible.
(2) Set up the reaction: Weigh out 10.0 g of potassium
bicarbonate and 2.23 g of GABA into the flask. Add the stir bar
and fill the flask slightly more than halfway with anhydrous
methanol. If a nitrogen tank is available, blow some N2 into the
flask above the methanol and then stopper. Begin vigorous stirring
on a magnetic stir plate, but without splashing. After 15 min, add
6.9 mL of methyl iodide to the reaction vessel by glass pipet.
(Caution: methyl iodide is toxic; wear gloves and perform this step
in a fume hood.) Stopper the flask and let stir for 2-3 days (40
and 66 h have produced similar yields).
Figure 2. Chemical structures of the TMAB labels. The net masses,
in daltons, are of the carbon chain, the carbonyl oxygen, and the
trimethyl ammonium group, minus the N-hydroxysuccinimide group
and the chloride ion (i.e., the mass difference in a peptide after
incorporating one of these compounds on a free N-terminal amine).
(3) Stop the reaction: Remove the stir bar from the solution.
Using a rotary evaporator, reduced pressure, and mild heating,
evaporate all methanol. Solvent distilled from the reaction may
contain residual methyl iodide; dispose of accordingly.
protocol intended for biochemists without formal training in
organic chemistry. Additional notes on the synthesis are provided
as Supporting Information. All of the procedures involving organic
solvents should be performed in a fume hood, and some of the
reagents are especially toxic (methyl iodide and dicyclohexylcar-
bodiimide). Proper safety equipment should be used to handle
these compounds and care should be taken to minimize exposure.
The procedures described below are identical for all four isotopic
tags; although the molecular weights and densities of the methyl
iodide vary, the differences are so small as to be negligible. The
use of CH3I results in the production of D0-TMAB-NHS
(Figure 2), which was previously referred to as H9-TMAB-NHS.22
The use of methyl iodide with three deuterium atoms in place of
the hydrogen atoms (CD3I) produces D9-TMAB-NHS, as previ-
ously described.22,23 The two new labels are produced from CH2DI
and CHD2I and result in the formation of D3-TMAB-NHS and D6-
TMAB-NHS, respectively (Figure 2).
(3-Carboxypropyl)trimethylammonium Chloride; Com-
mon Name: Trimethylammonium Butyric Acid Chloride
(TMAB Acid). Chemist Protocol. GABA (2.23 g, 21.6 mmol) and
potassium bicarbonate (10.0 g, 99.9 mmol) were combined in 250
mL of methanol under N2 with stirring. After 15 min, 6.9 mL of
methyl iodide (approximately 16 g, 110 mmol) was added. The
reaction was stirred for 66 h at ambient temperature. Then the
solvent was removed by evaporation under reduced pressure at
∼55 °C. The resulting white solid was washed four times with 50
mL of chloroform. The solid was then dissolved into 50 mL of
diluted concentrated HCl (3:1 water/acid), followed by removal
of bulk water by evaporation under reduced pressure under
moderate heat (∼60-65 °C). The resultant orange residue was
placed under high vacuum for further drying. After 2-3 h the
amber residue was extracted four times with 60 mL acetone. The
acetone was removed by evaporation under reduced pressure at
∼55 °C, yielding an oily amber residue. To this, 150 mL of THF
was added, and then the mixture was cooled to 0 °C for 0.5 h.
The resulting solid was filtered and washed further with THF,
(4) Wash and acidify the product: In a fume hood, pulverize
the white reaction product into a fine powder and add to a fritted
funnel, attached to sidearm flask connected to a vacuum line.
Wash the powder four times with 50 mL of chloroform. Add the
powder to a 250 mL round-bottom flask and remove the remaining
solvent under reduced pressure. Acidify the powder with 50 mL
of a 3:1 mixture of water/concentrated HCl. Agitate the flask or
stir while adding acid slowly. Remove the acid/water on a rotary
evaporator, with reduced pressure and heating to 60-65 °C. Then
place under high vacuum for further drying.
(5) Extract the product into acetone: Add 60 mL of anhydrous
acetone to the product, agitate to suspend all solids, then pour
into the fritted funnel/sidearm flask apparatus as before. Wash
the solid three more times with 60 mL of acetone. Discard the
solid. Add the acetone to the round-bottom flask, remove the
solvent by rotary evaporator as before, with mild heat (ap-
proximately 55 °C).
(6) Clean and dry the product: Slowly add 150 mL of anhydrous
THF to the product while mixing (swirling flask by hand). It may
be necessary to break up the oily product with spatula to yield a
solid. Stopper the flask and place flask in ice to allow further
precipitation. This precipitate is the reaction product (TMAB acid).
Filter the precipitate in a glass funnel, wash several times with
50-100 mL of anhydrous THF, and then dry under vacuum for
16 h. After weighing, if you will not proceed with the second step
(below), it is best to store it under nitrogen in a desiccator that is
cold and dark.
[3-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)-propyl]-trime-
thylammonium Chloride; Common Name: Trimethylammo-
nium Butyric Acid Chloride N-Hydroxysuccinimide Ester
(TMAB-NHS). Chemist Protocol. TMAB acid (2.02 g, 11.1 mmol),
the methylated amine, was dissolved into 250 mL of anhydrous
acetonitrile and cooled to 0 °C. NHS (2.18 g, 19.0 mmol, 1.7 equiv)
9300 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

was added with stirring, followed by dicyclohexylcarbodiimide
(4.30 g, 20.9 mmol, 1.9 equiv). The reaction was allowed to warm
to ambient temperature over 16 h. The reaction was cooled to 0
°C again at 16 h, and the stirring was terminated. The reaction
mixture was filtered, the solid was discarded, and the acetonitrile
was evaporated under reduced pressure. The compound was
slurried with 125 mL of THF and cooled to 0 °C for 1 h. The
resulting white solid was filtered and dried under vacuum. This
reaction typically yielded 2.72 g (9.74 mmol, 88% yield for the
second step) of TMAB-NHS.
Biologist Protocol. (1) Prepare the equipment: Dry a 500 mL
round-bottom flask, stopper, and stir bar in a vacuum oven, as
above. Set up the flask above the magnetic stirrer, as before, but
include a tray on top of the magnetic stirrer that can hold ice (do
not add ice yetssee below).
(2) Start the reaction: If you have higher or lower yields from
step 1 (formation of the TMAB acid), scale the following reaction
up or down as appropriate, being careful to maintain the same
proportion of all reagents. Weigh 2.02 g of TMAB acid and 2.18 g
of NHS and add to the flask with the stir bar. Add 250 mL of
anhydrous acetonitrile, stopper the flask, and stir to dissolve the
reagents at room temperature for approximately 15 min. Then,
add ice to the tray so that the acetonitrile solution cools while it
is stirring. Weigh out 4.30 g of dicyclohexylcarbodiimide and
dissolve in 43 mL of anhydrous acetonitrile at room temperature.
Add the dicyclohexylcarbodiimide/acetonitrile solution to the
flask, replace the stopper, and stir for 16 h. During this time, the
ice in the bath will melt and the reaction will warm to room
temperature.
(3) Remove the precipitate and isolate the product: Turn off
the stirrer and place the flask in an ice bath to allow the
dicyclohexylurea to precipitate. Filter off the precipitate using a
fritted funnel and wash the precipitate with 100 mL of acetonitrile.
Discard the precipitate; the desired reaction product is in the
acetonitrile. Place the acetonitrile solution into a round-bottom
flask and remove the solvent on a rotary evaporator under reduced
pressure, as before, yielding a brown oily residue.
(4) Clean and dry the product: Slowly add 125 mL of anhydrous
THF to the residue while swirling by hand. Place the flask in an
ice bath to allow a precipitate to form (usually 1-2 h). It may be
necessary to break up the oily product with a spatula to yield a
solid. Once formed, isolate this solid with a glass filter, washing
the residue with 50-100 mL of THF (wash until the compound
is nearly white). Dry the precipitate under vacuum for 16 h, then
weigh and place it in a storage vial. For long-term storage, it is
best to gently blow nitrogen into the tube, then tightly seal and
store it in a cold and dark desiccator.
Labeling of Mouse Brain Extract. Four mice (C57B6/J, 2-3
month old) were bred in the barrier facility of the Albert Einstein
College of Medicine. Each mouse was sacrificed by decapitation,
and the head was immediately irradiated in a conventional
microwave oven for 8 s to raise the brain temperature to 80 °C,
as described.29 This microwave irradiation step is necessary to
rapidly inactive proteases that can rapidly degrade cellular proteins
within the postmortem processing; these protein degradation
fragments overwhelm the signal from neuropeptides.29-31 In
addition, microwave irradiation inactivates proteases that degrade
neuropeptides, and the level of recovered neuropeptides is several-
fold higher in microwave-irradiated brain extracts than in nonmi-
crowaved tissue.29,31 After cooling, the mouse brain was isolated,
the olfactory bulb and cerebellum were removed, and the
remainder of the brain was frozen in dry ice and stored at -70
°C until peptide extraction.
Each mouse brain was sonicated for 50 pulses at 1 pulse/s in
1.0 mL of ice-cold water using an ultrasonic processor (W-380,
Ultrasonic Inc., Farmingdale, NY). The homogenate was incu-
bated in a 70 °C water bath for 20 min, cooled in an ice bath, and
acidified with 120 µL of 0.1 M HCl to a final concentration of 10
mM HCl. The homogenate was centrifuged at 13 000g for 30 min
at 4 °C, and the supernatant (peptide extract) was transferred to
a new low-retention tube. Aliquots (60 µL each) of the brain
peptide extract from each of four mice were pooled, mixed well,
and then split into four equal parts. Each part was neutralized to
pH 8 by 75 mM borate buffer, and the final pH 9.5 was reached
by addition of 1 M NaOH before the labeling reaction. For the
labeling reaction, 6.4 µL of 350 µg/µL D0-, D3-, D6-, or D9-TMAB-
NHS dissolved in DMSO was added separately to four peptide
extract sample tubes. After 10 min, an appropriate volume of 1.0
M NaOH was added to adjust the pH back to 9.5 and the tubes
were also incubated for 10 min. The addition of TMAB and NaOH
was repeated seven times, then the mixtures were incubated at
room temperature for another 1 h, after which 10 µL of 2.5 M
glycine was added into each labeling tube to quench the remaining
TMAB reagents. After labeling, the four TMAB-labeled samples
were combined and filtered through a Microcon YM-10 unit
(Millipore) to cut off proteins >10 kDa. To remove any labels
from Tyr residues in the peptides, the pH of filtrate was adjusted
to 9.0 and a total of 13.5 µL of 2.0 M hydroxylamine (in DMSO)
was added in three aliquots of 4.5 µL each, with an interval of 10
min between each addition. After desalting with a PepClean C-18
spin column (Pierce), peptides were eluted out of the column with
80 µL of 70% acetonitrile in 0.1% trifluoroacetic acid in water,
frozen, and concentrated to 20 µL in a vacuum centrifuge. Aliquots
of the peptide samples were stored at -70 °C until MS analysis.
LC/MS/MS. The peptide mixture was trapped and washed
on a PepMap C18 trapping column (5 µm, 100 Å, 300 µm i.d. × 5
mm, LC Packing, Marlton, NJ) using a Eldex MicroPro syringe
pumping system with 5% solvent B for 20 min at a flow rate of 4
µL/min. Then the peptides were separated on a Grace Vydac MS
C18 capillary column (3 µm, 100 Å, 75 µm i.d. × 150 mm, Hesperia,
CA) at 4 µL/min with a gradient elution 5% solvent B for 45 min,
then to 35% solvent B in 40 min, to 95% solvent B in the following
20 min. Solvent A was 2% acetonitrile in 0.1% formic acid. Solvent
B was 80% acetonitrile in 0.1% formic acid. Flow from the column
was directed to an API Q-Star Pulsar-i quadrupole time-of-flight
mass spectrometer (Applied Biosystems/MDS Sciex, Foster City,
CA). The nanoelectrospray ion source was operated in the positive
mode with a spray voltage of 2 kV. The information-dependent
acquisition mode was used with a 1 s survey scan and 2 s MS/
MS scan on the most intense ions in the MS survey scans. The
(30) Parkin, M. C.; Wei, H.; O’Callaghan, J. P.; Kennedy, R. T. Anal. Chem. 2005,
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Kennedy, R. T. Anal. Chem. 2006, 78, 4342–4351
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2005, 4, 1391–1405
.
.
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008 9301

collision energy for MS/MS (20-45 eV) was dynamically selected
based on the m/z value and charge state of the ion selected.
Data Analysis. The Mascot program (http://www.matrix-
science.com) was used to aid in the interpretation of tandem mass
spectra. In Mascot searches, fixed modifications were selected
as GIST-Quat, GIST-Quat:2(H)3, GIST-Quat:2(H)6, or GIST-Quat:
2(H)9 for D0-, D3-, D6-, or D9-TMAB-labeled peptides at the
N-terminus and Lys residues. (Note that this involved four Mascot
searches, one for each isotopic tag, because in the “fixed
modification” mode the different isotopes are mutually exclusive.)
Although Mascot searches identified a number of peptides, this
program was not able to identify some MS/MS spectra that were
of medium-high quality, primarily due to partial breakdown of the
TMAB tags. Those MS/MS spectra that could not be identified
by Mascot were interpreted manually. Also, manual reinterpreta-
tion of Mascot hits was done to verify the predicted peptide
sequence. The criteria for considering a peptide identified from
the MS/MS analysis were (i) a parent mass within 40 ppm
(preferably 20 ppm) of the theoretical mass; (ii) the observed
number of TMAB tags on the peptide matched the predicted
number of free amines available (i.e., Lys residue and N-terminus);
(iii) the observed charge state(s) of the peptide was consistent
with the expected number of positive charges; (iv) 80% or more
of the major fragments observed in MS/MS matched predicted
fragments (minimum of five matches). Representative spectra of
the peptides are included in the Supporting Information ac-
companying this paper. Mascot search results are shown for some
peptides, and manual interpretations are also provided for those
peptides that were not found by Mascot.
The intensity of each TMAB-labeled isotopic peak was deter-
mined by measuring peak heights of the monoisotopic peak and
the additional peaks containing one and two atoms of 13C. As an
alternative to peak height, it is possible to quantify by measuring
peak area when the peaks are well separated, but this approach
is more difficult to use when there is peak overlap, and so for the
present study, all measurements were made using peak height.
When the peaks for the D0, D3, D6, and D9 forms of each peptide
are well separated (Figure 3A), the quantitation was performed
by simply measuring the peak heights. However, for those
peptides with overlapping peaks it was necessary to calculate the
theoretical peak intensity of the 13C isotope-containing peaks
(Figure 3B). This calculation was performed using the Protein
Prospector program (http://prospector.ucsf.edu/). The baseline
was then adjusted for the D3, D6, and D9 peaks based on this
theoretical distribution, as shown in Figure 3B.
To measure the reproducibility between the four TMAB
labeling reagents, the peak heights for each of the TMAB labels
was compared to the average peak height of the other three peaks.
For example, the following ratios were determined: the D0-TMAB
peak versus the average of the D3, D6, and D9 peaks; the D3-
TMAB peak versus the average of the D0, D6, and D9 peaks; the
D6 peak versus the average of the D0, D3, and D9 peaks; the D9
peak versus the average of the D0, D3, and D6 peaks. The
standard deviation of all four ratio measurements for each peptide
was determined using Excel.
Figure 3. MS spectra of peptides labeled with the isotopic TMAB tags
and analyzed by LC/MS. (A) Example of a peptide that displays baseline
resolution between each isotopic tag. The peptide was identified by MS/
MS analysis as neuromedin N (KIPYIL) which incorporated two isotopic
tags. The indicated monoisotopic m/z values represent the 2+ ion. (B)
Example of a peptide with relatively smaller mass differences between
isotopic peaks and which therefore shows substantial peak overlap. This
peptide was identified by MS/MS analysis as a chromogranin B-derived
peptide corresponding to residues 438-454 (LLDEGHYPVRESPIDTA).
The indicated monoisotopic m/z values represent the 3+ ion that
incorporated one isotopic tag (on the N-terminus) and protons on the
His and Arg residues. To determine the relative abundance of each
isotopic form, it is necessary to subtract the contribution from the 13C-
containing peptides of the lower m/z values. For this, Protein Prospector
was used to determine the natural isotope distribution of this peptide,
and the baseline of each peak was then adjusted to account for the
contribution from the 13C-containing isotope of the lower m/z peak, as
shown in panel B.
RESULTS
The synthetic scheme for the production of the TMAB labels
(Figure 1) is a simple two-step reaction that can be performed by
nonchemists with relatively simple equipment that, except for the
rotary evaporator, is standard in biochemistry laboratories. The
9302 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

first step involves the reaction of methyl iodide with 4-aminobu-
tyric acid (also called γ-aminobutyric acid, or GABA). The yield
of the reaction product is high if care is taken to avoid the
introduction of water into the reaction by drying the reagents and
using anhydrous solvents. Typical yields of the first reaction step
are 60-70%. The reaction with regular methyl iodide (CH3I) and
fully deuterated methyl iodide (CD3I) has been previously
described.22,23 In the present study, the reaction with CH2DI and
CHD2I was performed and found to proceed with the same overall
yield as for the other compounds. The product of this first reaction
was then reacted with dicyclohexylcarbodiimide and NHS to form
the active ester (Figure 1). The yield of this second step was
typically 80-90%, for an overall yield of 50-60%. The active NHS
ester is stable for over 1 year when stored under anhydrous
conditions.
The four isotopic tags differ in mass by 3 Da, and after reaction
with the amine group of a peptide they increase the mass by
129-138 Da per label incorporated (Figure 2). To test the labels
under typical experimental conditions, the peptide extract from
several mouse brains was divided into equal parts and labeled
with the four isotopic tags. After quenching, the reactions were
pooled, treated with hydroxylamine to remove the tags from Tyr
residues, and then desalted and analyzed by LC/MS. Representa-
tive data are shown in Figure 3. For all peptides identified in the
present study, every free N-terminal amine and Lys side chain
was labeled with the TMAB reagent, indicating that the labeling
efficiency was close to 100% (Table 1). In most cases where
multiple tags were incorporated into the peptide (i.e., when there
were Lys residues), the 3 Da difference between the mass of each
tag was easily resolved upon analysis of the data (Figure 3A). For
those peptides that did not contain Lys residues and therefore
incorporated only a single tag on the N-terminal amine, the
separation of the individual peaks was less pronounced (Figure
3B). In these cases, determination of the peak heights attributed
to the D3, D6, and D9 peaks required calculation of the distribu-
tion of 13C isotopes in the peptide and subtraction of their
contributions to the overall intensity (Figure 3B).
the parent ion. In this example, the parent MH2+ ion (detected at
820.97) undergoes neutral loss of one and two trimethylamine
groups (i.e., -59 and -118 Da for the D0 form of the TMAB
labels) to produce the 2+ ions at 791.43 and 761.89, respectively
(Figure 5A). All of the daughter ions have completely lost the
trimethylamine groups, which is the state considered by Mascot,
and so this program correctly identified the peptide. However,
other peptides not identified by Mascot showed beautiful sequence
ladders that perfectly matched known peptides present in brain;
for example, the proSAAS-derived peptide shown in Figure 5B.
In this example, the peptide fragments do not lose the trimethy-
lamine group (68 Da for this D9-TMAB label) and therefore are
not considered by the Mascot program. Because every major MS/
MS fragment matches a predicted fragment from this proSAAS-
derived peptide, and Mascot failed to identify a better match, it is
highly likely that this assignment is correct. Additional notes on
the interpretation of spectra and the complication of the partial
breakdown of the TMAB labels is provided as Supporting
Information.
Altogether, 107 peptides were identified in the present study
(Table 1). The Mascot results and/or manual interpretation
are provided in the Supporting Information. Of these 107
peptides, 72 are derived from secretory pathway proteins and
the other 35 are fragments of cellular proteins. The secretory
pathway protein fragments generally represent either known
neuropeptides or other peptides that are predicted to be
produced based on the presence of prohormone convertase
cleavage sites flanking the peptide. Most of these secretory
pathway protein fragments have been previously identified in
peptidomics analyses. Some, but not all, of the cellular protein
fragments have been previously reported in peptidomics stud-
ies. The majority of the observed peptides, both secretory
pathway and cellular, represent unmodified peptides (except
for the proteolytic cleavages). However, some show post-
translational modifications beyond the proteolysis required for
their production. These post-translational modifications include
C-terminal amidation, N-terminal acetylation or pyroglutamy-
lation, phosphorylation of Ser, oxidation of Met, and two
additional modifications; one a +25 Da modification (cyanyla-
tion) on the N-terminal Cys of the ubiquinol-cytochrome c
reductase hinge protein fragment of 1324.75 Da, and the other
the loss of 36 Da (two waters) from the proSAAS fragment of
1296.68 Da (Table 1).
For isotopic labels to be optimal for quantification applications,
it is important that the labeled peptides coelute on the HPLC
columns used for LC/MS. Peptides labeled with all four of the
isotopic labels tested in the present study coelute from reversed-
phase columns (Figure 4). This is true both for peptides with a
single isotopic tag (Figure 4, right panels) as well as those with
multiple tags (Figure 4, left panels). In all cases, the elute time
for each of the isotopic peaks was identical.
Another important feature of isotopic tags is that they allow
for the conclusive identification of the peptide from MS/MS data.
The TMAB labels undergo partial breakdown upon CID that can
complicate the spectra and lead to lower scores on Mascot and
other search programs. However, manual interpretation of the
spectra with knowledge of the typical breakdown patterns can
greatly increase the confidence in the assignments and/or
eliminate false positives. For example, Figure 5A shows the MS/
MS spectrum of a peptide identified by Mascot, with many
matches to predicted b- or y-series ions. However, the two major
ions in the spectrum (791.43 and 761.89) were unmatched by
Mascot. This program only considers partial breakdown of the
TMAB tags in the MS/MS mode and not partial breakdown of
For the vast majority of the peptides (>90%), the relative
intensity of each isotopic peak was within 20% of the average
intensity of the three other peaks (Table 1 and Figure 6). Only
two peptides showed spectra with one of the peaks substantially
different than the other three peaks; the prodynorphin peptide of
1027.55 Da, and the ubiquinol-cytochrome c reductase hinge
protein fragment of 2450.15 Da (Table 1). The reason for this
variability for these two peptides is not clear. The standard
deviation of the relative peak intensities was within 0.20 for all
but six of the 107 peptides detected in the present study. Thus,
the four isotopic labels are able to provide quantitative data on
the levels of a large number of peptides in a single analysis.
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9304 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

Analytical Chemistry, Vol. 80, No. 23, December 1, 2008 9305

Figure 4. Four isotopically labeled forms of each peptide coelute
from a reversed-phase LC column. Left panels represent chromog-
ranin B (64-86), and the right panels represent chromogranin B
(438-454). The relative intensity for m/z values representing each
isotopic form are plotted vs elute time in minutes.
DISCUSSION
Although the TMAB labels were developed several years ago
by Regnier’s group,23 use of these labels has been limited.22,28,29,32
This is presumably due to the lack of a commercial supplier for
these compounds. However, the synthesis is very simple and can
be done by a nonchemist with respectable yields. For example,
the protocol described in the Materials and Methods section was
performed by a biologist with no experience in synthetic organic
chemistry and an overall yield of 45% was achieved. The TMAB-
NHS labels are produced by convenient “dump-and-stir” reactions
in two steps. The first step is the reductive alkylation of an amine
by methyl iodide, and the second is a standard carbodiimide-
catalyzed activation of a carboxylic acid to form a reactive ester
(i.e., the NHS reagent that will subsequently react with amines
on the peptides in the biological sample). To increase the yield
of the synthetic reaction it is important to start with anhydrous
reagents, solvents, and glassware and to protect the solvents from
ambient atmospheric conditions. Additional tips to improve the
yield for the synthesis are included as Supporting Information.
This reaction is easily scaled up or down, and the product will
keep for years if protected properly.
Previously, an advantage of the iTRAQ reagents was their
availability in four versions, allowing for multiple comparisons in
a single LC/MS run. With the synthesis of the D3 and D6 isotopic
forms of the TMAB labels, it is now possible to perform
multivariate comparisons using the TMAB labels. In addition to
the four TMAB labels described in the present manuscript, a fifth
TMAB isotopic label is possible using 13CD3I in the first step.
This would result in a compound that is 3 Da heavier than the
D9-TMAB reagent, extending the series by one additional label.
Although the 3 Da difference between each TMAB isotopic label
is not sufficient for the baseline separation of each isotopic form
of some peptides that incorporate only a single isotopic tag (see
(32) Che, F.-Y.; Biswas, R.; Fricker, L. D. J. Mass Spectrom. 2005, 40, 227–237
.
9306 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

Figure 5. Representative MS/MS spectra. (A) MS/MS of the proenkephalin-derived peptide SPQLEDEAKELQ, which incorporated two D0-
TMAB tags. Note the complete loss of trimethylamine from the observed a-, b-, and y-series ions. The spectrum also shows an MH2+ peak for
the intact peptide and stronger peaks corresponding to the loss of 59 and 118 Da (i.e., 2 × 59); these represent neutral loss of one and two
trimethylamines from the parent ion. (B) MS/MS of D9-TMAB-labeled proSAAS-derived peptide SLSAASAPLVETSTPL. For this peptide, many
of the trimethylamine groups are not lost in the collision-induced dissociation, so most of the b-series ions are indicated as +68 (for D9-TMAB-
labeled peptide). In this example, partial breakdown of tags leads to inconclusive results upon Mascot database searches, so manual interpretation
is required.
Figure 3), it is possible to deconvolute the data by calculating
the theoretical isotopic distribution of 13C. This allows for the
appropriate baseline to be subtracted from the D3, D6, and D9
peaks, providing more accurate quantification of the peak intensities.
As expected on the basis of the previous results with the D0-
TMAB (formerly called H9-TMAB) and the D9-TMAB re-
agents,22,23 all isotopic forms of the TMAB-labeled peptides coelute
from reversed-phase HPLC columns (see Figure 4). This is an
important requirement for isotopic tags because the relative
intensities of peptides are more accurate when comparing the
same MS scans than when comparing peptides in distinct scans.
This has been found to be a problem with many isotopic labeling
reagents that use deuterium and has led to the development of
13C- and/or 15N-based labeling reagents.15,33 These other isotopes
do not affect elution from the reversed-phase HPLC columns, and
so the heavy and light forms coelute. However, 13C- and 15N-
labeled compounds are much more expensive than deuterated
compounds. The design of the TMAB reagents by Regnier’s
group23 was based on the theory that the deuterium in the heavy
form of the label would not interact with the reversed-phase matrix
due to the adjacent quaternary amine. This elegant idea combines
the advantages of the deuterium labels (low cost, easy synthesis,
and labels that differ by 3 Da or more) and eliminates the major
limitation (labeled peptides that do not coelute on HPLC).
However, the quaternary amine causes the label to be unstable
in many different MS applications (discussed below).
Although in theory each peptide labeled with the four
isotopic TMAB reagents should appear in precise 1:1:1:1 ratios,
this was only the case for some of the peptides, and most
showed a slight deviation of one or more of the peaks. The
data shown in Figure 3 are truly representative; for example,
(33) Julka, S.; Regnier, F. E. J. Proteome Res. 2004, 3, 350–363
.
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008 9307

labeled peptide when analyzed in MS mode, with virtually no
breakdown of the label. However, after CID for MS/MS sequenc-
ing, the labeled peptides usually decompose and lose trimethy-
lamine (which is neutral and therefore not detectable by MS).
The positive charge is retained on the label attached to the peptide.
Because the differential isotopes are usually lost from the
molecule, the MS/MS results for each of the various isotopic
forms are generally identical. However, for some peptides the
breakdown of the TMAB label is not complete and the trimethy-
lamine moiety remains attached to the peptide. This is especially
a problem for peptides with multiple tags. When MS/MS results
are obtained for multiple isotopic forms of a particular peptide, it
is easy to spot these incompletely degraded TMAB-labeled
fragments; they are the ones that differ in m/z between the spectra
for each of the isotopic forms.
Due to the partial breakdown of the TMAB labels in CID and
MS/MS, Mascot scores are often lower than those considered to
be acceptable for conclusive identification of a peptide. However,
manual reinterpretation of these spectra is relatively straightfor-
ward, and if the majority of the peaks can be assigned (taking
into account the partial breakdown of the TMAB labels, as well
as other possible ions such as a-ions, loss of water or ammonia
from b- or y-ions, and internal fragments) then it is highly likely
that the identification is correct.
The majority of the peptides identified in the present study
are previously identified secretory pathway peptides. Many result
from cleavages at known processing sites, often pairs of basic
amino acids, by an endopeptidase followed by a carboxypeptidase.
Some of the peptides detected in the present study have
undergone additional post-translational modifications, including
phosphorylation of Ser, oxidation of Met, and amidation of the
C-terminus (by oxidation of a Gly on the C-terminus of the
processing intermediate). One of the peptides, a 1296.68 Da
fragment of the peptide named little SAAS, appears in a form 36
Da lighter than the theoretical peptide. The loss of 36 occurs on
the C-terminal five amino acids (sequence VETST) based on
analysis of the b- and y-ions. This peptide presumably has
undergone loss of two water molecules; a peptide 18 Da lighter
than the theoretical peptide was detected in some LC/MS
analyses, and although this other peptide has not been sequenced,
it likely reflects the SAAS-derived peptide with the loss of one
water molecule. In addition to the peptides derived from secretory
pathway proteins, a number of peptides derived from cellular
proteins (i.e., cytoplasmic, nuclear, lysosomal, mitochondrial) were
detected in the present study. Some of these peptides have been
reported in previous peptidomics studies. Interestingly, one of
these peptides, a fragment of ubiquinol-cytochrome c reductase
contains a +25 Da modification on the N-terminal Cys residue;
this corresponds to the mass difference of a cyano group.
Figure 6. Distribution of peak ratios of the 107 peptides detected in
the analysis of mouse brain. The peak-to-peak ratios of each peptide
fragment were measured, and the individual peak intensity for each
isotopically labeled form was compared to the average peak intensity
of the other three isotopically labeled forms of the peptide (see Table
1, columns 8-11). Then, the number of peptides for each ratio was
plotted vs the observed ratio. The theoretical ratio is 1.00, and nearly
all of the isotopically labeled forms of each peptide showed ratios
within 20% of this theoretical ratio.
the peptide in Figure 3B (the chromogranin B-derived peptide
LLDEGHYPVRESPIDTA) has peak heights that are among the
most variable of all peptides found in the present study
(standard deviation ) 0.18; see Table 1). Most peptides showed
less variability than this peptide. In this experiment, variability
is not due to animal-to-animal variation because extracts from
several animals were pooled prior to labeling. In those cases
where one peak is higher than the others, it is possible that
this is due to the coelution of another peptide with similar m/z
as one of the peaks. But, in cases where one peak is lower
than the other three, this is less likely (it would require three
coeluting peaks of precisely the same mass and charge). Still,
most peptides showed a ratio that was very close to the
theoretical 1:1:1:1 ratio, and those that did not were still within
the normal animal-to-animal variations seen with typical experi-
ments using the D0 and D9 isotopic tags.34-37 Thus, the small
background variation observed with the labels in the present
study will be negligible for most experiments. Furthermore,
performing experiments with 3-4 replicates using different tag
combinations for each replicate will greatly reduce the potential
error due to minor fluctuations in individual isotopic peaks.
In matrix-assisted laser desorption ionization time-of-flight MS,
the TMAB labels lose trimethylamine from a substantial fraction
of the tagged peptides, thus reducing the level of the isotopically
labeled peptide and complicating the spectra with additional peaks.
Ion trap mass spectrometers with electrospray ionization have not
been successfully used with the TMAB-labeled peptides.16 Best
results have been observed with electrospray ionization quadru-
pole time-of-flight mass spectrometers (ESI Q-TOF MS). Compa-
rable results were obtained with TMAB-labeled peptides analyzed
on ESI Q-TOF MS instruments manufactured by several different
companies (MDS Sciex/Applied Biosystems; Waters/MicroMass).
All ESI Q-TOF MS instruments tested show the intact TMAB-
CONCLUSIONS
The major finding of the present study is that four isotopic
forms of the TMAB labels can be readily synthesized and the
labeled peptides coelute from reversed-phase HPLC columns and
show the expected 1:1:1:1 ratio. The development of four labels
allows for a large variety of studies that are either not possible or
are less efficient than studies with only two isotopic forms of the
labels. For example, with four labels it is possible to include
duplicates of control and treated groups in the same study, thus
(34) Che, F.-Y.; Yuan, Q.; Kalinina, E.; Fricker, L. D. J. Biol. Chem. 2005, 280,
4451–4461
(35) Che, F.-Y.; Vathy, I.; Fricker, L. D. J. Mol. Neurosci. 2006, 28, 265–275
(36) Lim, J.; Berezniuk, I.; Che, F.-Y.; Parikh, R.; Biswas, R.; Pan, H.; Fricker,
L. D. J. Neurochem. 2006, 96, 1169–1181
(37) Pan, H.; Che, F. Y.; Peng, B.; Steiner, D. F.; Pintar, J. E.; Fricker, L. D.
J. Neurochem. 2006, 98, 1763–1777
.
.
.
.
9308 Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

providing a reflection on the variation between duplicates in a
single LC/MS run. Alternatively, it is possible to compare several
different treatment groups in the same experiment. Although the
applications described in this study involve peptidomics analyses,
these TMAB labels should be applicable to proteomic studies
much like the iTRAQ reagents. But, because the TMAB labels
do not require MS/MS for the quantitation, it is possible to obtain
quantitative data on any peptide detected in the MS mode. Thus,
these TMAB labels should have broad applications for a variety
of projects.
Cardoene for testing the “biochemist” version of the synthetic
scheme and offering comments. Mass spectrometry was per-
formed in the Laboratory for Macromolecular Analysis and
Proteomics of the Albert Einstein College of Medicine, Dr. Ruth
Angeletti, director. The first two authors contributed equally to
this work.
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGMENT
Received for review August 6, 2008. Accepted October 7,
2008.
This work was supported by NIH Grants DA-04494 and DK-
51271 to L.D.F. Thanks to Iryna Berezniuk for providing the
mouse brain extract used in the present study and to Dries
AC801654H
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008 9309