A novel pyrimidine-based stable-isotope labeling reagent and its application to quantitative analysis using matrix-assisted laser desorption/ionization mass spectrometry

Article abstract of DOI:10.1002/jms.1260

As an extension of our previous work, a novel pyrimidine-based stable-isotope labeling reagent, [d₀]-/[d₆]-4,6-dimethoxy- 2-(methylsulfonyl)pyrimidine (DMMSP), was developed for comparative quantification of proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our one-step labeling strategy combines several desirable properties such as cysteine-specific labeling, signal amplification and direct analysis with minimum sample handling. All these features not only allow easy interpretation for protein identification and quantification but also ensure rapid and sensitive progression to MS analysis. Using cysteine, Cys-containing peptide, and lysozyme digest as model samples, the labeling methodology was established and the following pilot application for quantitative analysis was accomplished with high confidence, accuracy, efficiency, and reproducibility. The application of DMMSP-labeling strategy is expected to provide a powerful new tool for comparative proteome research, especially for the analysis of low-abundance proteins. Copyright

Full text of DOI:10.1002/jms.1260

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2007; 42: 1514–1521
Published online 6 July 2007 in Wiley InterScience
(
www.interscience.wiley.com) DOI: 10.1002/jms.1260
A novel pyrimidine-based stable-isotope labeling
reagent and its application to quantitative analysis
using matrix-assisted laser desorption/ionization mass
spectrometry
∗
Jing Zhang, Li Zhang, Ying Zhou and Yin-Long Guo
Shanghai Mass Spectrometry Center, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
Received 9 March 2007; Accepted 23 May 2007
As an extension of our previous work, a novel pyrimidine-based stable-isotope labeling reagent, [d
0 6
]-/[d ]-
4
,6-dimethoxy-2-(methylsulfonyl)pyrimidine (DMMSP), was developed for comparative quantification of
proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS). Our one-step labeling strategy combines several desirable properties such as cysteine-specific
labeling, signal amplification and direct analysis with minimum sample handling. All these features
not only allow easy interpretation for protein identification and quantification but also ensure rapid
and sensitive progression to MS analysis. Using cysteine, Cys-containing peptide, and lysozyme digest
as model samples, the labeling methodology was established and the following pilot application for
quantitative analysis was accomplished with high confidence, accuracy, efficiency, and reproducibility.
The application of DMMSP-labeling strategy is expected to provide a powerful new tool for comparative
proteome research, especially for the analysis of low-abundance proteins. Copyright  2007 John Wiley &
Sons, Ltd.
KEYWORDS: 4,6-dimethoxy-2-(methylsulfonyl)pyrimidine; stable-isotope labeling; quantification; proteomics; MALDI-TOF
MS
INTRODUCTION
and the concurrent identification of proteins for proteomics
research.
Proteomics1,2 as a tool for comprehensive analysis of proteins
has been used for protein expression, localization, post-
translational modification, and protein–protein interaction.
Since proteome describes the immense dynamic process that
Stable isotope coding can already be performed during
11,12
cell culture, either by growth on isotope-labeled media
or by supplementing growth media with labeled amino
13–15
acids.
This strategy has the advantage that all further
3
occurs in a living organism, the quantitative analysis of
sample preparation steps can be performed after combining
the differentially labeled samples, thereby minimizing effects
global protein levels, termed ‘quantitative proteomics’, is
important in understanding the molecular function of each
protein component and is expected to provide insights into
molecular mechanisms of various biological processes and
16
of parallel sample processing. However, such metabolic
labeling strategy is sometimes unsuitable for many types of
samples (body fluids, human tissue, etc.), and so most of
the labeling techniques were realized through incorporation
of the stable isotope tags onto selected sites on peptides
via chemical reactions. It includes isotopic labeling of target
peptides at their N-/C-terminal or on specific amino acid
4
systems.
The two primary techniques, two-dimensional gel elec-
trophoresis (2-DE)-based methods and mass spectrometry
5
(
MS)-based methods with a stable isotope as the internal
6
–8
standard,
have proven successful for relative quantifica-
1
7–24
residues, such as cysteine, lysine, tryptophan, etc.
The
tion in proteomics. While gel-based techniques suffer from
limitations in protein coverage, sensitivity, dynamic range,
and precision of the measurements,
application of stable-isotope labeling followed by MS analy-
sis has emerged as a powerful technology for quantification
prototypical method to generate quantitative protein profiles
1
7
is the isotope-coded affinity tag (ICAT) reagent method.
9
,10
more recently, the
Since its advent, the ICAT method has been widely applied
to the comparison of the relative quantity of proteins
between samples (i.e. control vs perturbation) in a variety
2
5–29
30,31
of species including the cultured cells,
tissues,
and
3
2
body fluids.
In addition, several improvements have been made
for the later versions of ICAT reagents,
Ł
Correspondence to: Yin-Long Guo, Shanghai Mass Spectrometry
Center, Shanghai Institute of Organic Chemistry, Chinese Academy
of Sciences, Shanghai 200032, China. E-mail: ylguo@mail.sioc.ac.cn
2
0,21
and other
Copyright  2007 John Wiley & Sons, Ltd.

Pyrimidine-based stable-isotope labeling reagent
1515
different types of labeling reagents19,22–24 have also been
developed. For example, our group previously described a
solid-phase approach that uses a novel class of chemically
modified iodo-containing resins. The capture-and-release
process introduces stable isotope tags at cysteine residues
and isolates the tagged peptides. Also, the method is
demonstrated to have good ability to identify and quantify
(TFA, ½ 99.5%) were from Fluka. Acetonitrile and methanol
were both of HPLC grade and from Fisher Scientific (Fair
Lawn, NJ) and Merk KGaA (Darmstadt, Germany), respec-
tively. Deionized water was produced by a Direct-Q water
purification system (Millipore, ELPaso, TX). Other chemicals
were all of analytical grade without further purification.
3
3
Syntheses of [d ]- and [d ]-DMMSP
proteins in two samples.
0
6
Another alternatives is an imidazole-based reagent,34
The synthetic methods of the ‘light’ and ‘heavy’ forms were
3
6–40
based partly on previous published papers.
,6-Dichloro-2-(methylthio)pyrimidine
.5 mmol), dissolved in 10 ml of anhydrous methanol, was
2
-methoxy-4,5-dihydro-1H- imidazole, with specificity to-
4
(500 mg,
ward the lysine residue. The introduction of imidazol-2-
yl moiety enhanced the ionization efficiencies of lysine-
containing peptides, resulting in greater sequence coverage.
Additionally, this reactive imidazole reagent localizes the
charge at the C-terminal end of the peptides, resulting
in a highly simplified MS/MS spectrum consisting almost
exclusively of y-ions. This suggested that the employment of
nitrogenous heterocyclic rings would be advantageous for
peptide identification by MS.
2
added dropwise to a solution of 200 mg of sodium metal
in 20 ml of anhydrous methanol. (For preparation of the
[d ]-product, methanol-d was used instead of anhydrous
6 4
methanol.) The reaction mixture was stirred overnight
at 25 °C, then filtered and concentrated in vacuo. The
residue was taken up in 10 ml of water and extracted
with dichloromethane (3 ð 5 ml). The combined extracts
were dried over sodium sulfate and concentrated in vacuo,
3
5
As we formerly reported, 4,6-dimethoxy-2-(methylsul
fonyl)pyrimidine (DMMSP) has been applied as a new type
of protein modification reagent with high reactivity and
confirmed to have the ability to enhance the MS signals of
targeted molecules owing to the introduction of pyrimidine
ring. Furthermore, the lysozyme after DMMSP modifica-
tion retained its enzymatic activity with the synchronous
enhancement of stability, indicating that the DMMSP mod-
ification is a mild process and might not affect the active
region of lysozyme.
giving the crude product [d
methylthio)pyrimidine.
6
To a solution of [d ]- or [d ]-4,6-dimethoxy-2-(methylthio)
0 6
]- or [d ]-4,6-dimethoxy-2-
(
0
pyrimidine (400 mg, 2.1 mmol) in 40 ml of anhydrous
dichloromethane was added mCPBA (1.4 g, 8 mmol). The
reaction mixture was stirred for 5 h at 30 °C and quenched
with saturated aqueous sodium bisulfite. The organic layer
was washed with saturated aqueous sodium bicarbonate
(
3 ð 20 ml). The aqueous layers were combined and extracted
with dichloromethane (3 ð 40 ml). The organic layers were
combined, dried over magnesium sulfate and concentrated
to give the crude product. The residue was subjected to
column chromatography (silica gel, ethyl acetate/hexanes
In this study, the use of DMMSP is extended to a novel
pyrimidine-based stable-isotope labeling reagent [d
]-4,6-dimethoxy-2-(methylsufonyl)pyrimidine ([d
0
]- and
]-/[d ]-
[
d
6
0
6
DMMSP) for quantitative analysis. In our labeling strategy,
the combination of cysteine-specific labeling, signal enhance-
ment, and direct matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS) analy-
sis makes the identification and quantification more simple,
efficient, and reliable. We studied the overall feasibility and
validity of our own methodology for the labeling strat-
egy and investigated a pilot application for comparative
quantification by MALDI-TOF MS.
1
: 3), affording [d
solid.
The structure of [d
H-NMR (CDCl
, 300 MHz): υ D 6.19 (s, 1H), 4.05 (s, 6H),
0 6
]- or [d ]-DMMSP (400 mg, 67%) as white
0
]-DMMSP was confirmed as follows:
1
3
ꢁ
1
3
1
.33 (s, 3H); IR (neat, cm ): 1591, 1315, 1262, 1226, 1141,
067, 966, 761. The structure of [d
6
]-DMMSP was confirmed
1
as follows: H-NMR (CDCl
3
, 300 MHz): υ D 6.19 (s, 1H), 3.34
1
3
(s, 3H); C-NMR (CDCl
3
, 75 MHz): υ D 171.9, 164.4, 93.1,
ꢁ
1
6
9
C
3.7, 38.8; IR (neat, cm ): 1595, 1311, 1280, 1209, 1148, 1089,
CÐ
56, 763; MS (EI): m/z 224 (M ); HR MS (EI): m/z calcd For
CÐ
EXPERIMENTAL
7
H
4
D
6
N
2
O
4
S ꢀM ꢁ 224.0738, found: 224.0741.
Materials and reagents
Derivatization of amino acids with [d ]-DMMSP
The amino acid or their mixture (1 µmol) was dissolved in
0
4
,6-Dichloro-2-(methylthio)pyrimidine
(98%),
3-chloro
peroxybenzonic acid (mCPBA, ꢀ 77%) and ˛-cyano-4-
5
00 µl of 30 mmol/l ammonium bicarbonate buffer solution
pH 8.0–8.2). A solution of acetic acid was used to adjust the
pH to 6.5. To the solution, 5 µmol of the [d ]-DMMSP was
added and the reaction mixture was incubated at 37 C for
h.
hydroxycinnamic acid (CHCA, 99%) were from Aldrich.
(
4
Methanol-d (99.8 atom % D) was from Cambridge Iso-
0
tope Laboratories, Inc. (Boston, MA). Amino acids such
as lysine, tyrosine, cysteine, and proline were all from
Shanghai Bio Life Science & Technology Co. Ltd. (Shang-
°
2
hai, China). Peptides including His-Cys-Lys-Phe-Trp-Trp
Derivatization of peptides with [d ]-DMMSP
0
1
(
HW-6), angiotensin I human acetate hydrate (DL-10), [Glu ]-
The peptide or their mixture (1 µmol) was dissolved in 500 µl
of 30 mmol/l ammonium bicarbonate buffer solution (pH
8.0–8.2). A 2 µmol amount of DTT was added, and then a
solution of acetic acid was used to adjust the pH to 6.5. To
fibrinopeptide B human (ER-14) and neurotensin (EL-13)
were all from Sigma (St. Louis, MO). Proteins such as
lysozyme from chicken egg white were also from Sigma
(
St. Louis, MO). Trypsin (TPCK-treated) from bovine pan-
the solution, 5 µmol of the [d
reaction mixture was incubated at 37 °C for 2 h.
0
]-DMMSP was added and the
creas, dithiothreitol (DTT, ½ 99.0%) and trifluoroacetic acid
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 1514–1521
DOI: 10.1002/jms

1516
J. Zhang et al.
Labeling methods of cysteine and Cys-containing
peptide for comparative analysis
Two cysteine solutions with different concentrations (1 µmol
and 1.5 µmol of cysteine dissolved in 500 µl of 30 mmol/l
ammonium bicarbonate buffer solution) were treated with
RESULTS AND DISCUSSION
DMMSP labeling strategy
In our previous work, DMMSP was applied as a new class
of protein modification reagent, which presented excellent
reactivity towards various nucleophilic groups such as
amino, phenol, or mercapto groups in a buffer solution
of pH 8.5.
Recently, we have discovered that the reagent can
specifically react with the sulphydryl groups of cysteine
residues in an acidified reaction system (Fig. 1), based on
which we developed a novel stable-isotope labeling strategy
for quantitative protein analysis.
3
5
5
0 6
µmol of [d ]- and [d ]-DMMSP after the solutions acidified
to pH 6.5. After 2 h reaction, equal volumes of the solutions
were combined and the diluted mixture was subjected to
MS analysis. Similarly, two Cys-containing peptide (HW-6)
solutions with concentrations of 2 µmol/ml and 3 µmol/ml
were subjected to the same process in the presence of DTT
according to the method described above.
The stable-isotope labeling reagent (Fig. 2) contains the
following subunits: (1) a reactive leaving group with high
specificity and reactivity toward sulphydryl groups, which
is one of the most nucleophilic functional groups within
the peptide sequences; (2) a pyrimidine ring to facilitate the
ionization process in the mass spectrometer, resulting in
the increase of the MS signal; and (3) two methoxyl tags
that can easily incorporate stable isotopes, which can be
differentiated by MS.
Methods of lysozyme derivatization and
comparative analysis
The protein (lysozyme) (1 mg) was dissolved in 500 µl of
3
8
0 mM NH
4 3
HCO buffer solution containing 8 M urea (pH
.0–8.2). To the solution was added a 2 µmol amount of
35
DTT, and the mixture was incubated at 37 °C for 6 h. Then,
the solution was diluted to 1 ml with 30 mM NH HCO
buffer solution (pH 8.0–8.2). The digestion was performed
overnight with a trypsin/protein ratio of 1: 50 (w/w) at 37°C.
4
3
The DMMSP labeling strategy is outlined in Fig. 3.
Two protein samples, control and test, are proteolyzed
The resultant solution was further diluted to 2 ml with
3
0 mM NH
solution of acetic acid was used to adjust the pH to 6.5. To
the solution was added 5 µmol of the [d ]-DMMSP and the
4 3
HCO buffer solution (pH 8.0–8.2), and then a
0
reaction mixture was incubated at 37 °C for 6 h.
For quantification, two lysozyme samples with different
concentrations (1.8 and 2 mg/ml) were separately denatured,
reduced, and tryptic-digested followed by the further
0 6
differential labeling with [d ]-DMMSP and [d ]-DMMSP.
The conditions were identical with those listed above. After
combining equal volumes of sample solutions, the diluted
mixture was subjected to a MS-based comparative analysis.
Mass spectrometry
MALDI-TOF MS analyses were performed using a Voyager
DE-STR mass spectrometer (Applied Biosystems, Framing-
ham, USA) equipped with a 337 nm pulsed nitrogen laser. All
the spectra were acquired in positive reflectron mode at an
accelerating voltage of 20 KV. A total of 200 laser shots were
acquired and signal-averaged per mass spectrum. Instru-
ment control and data processing were maintained with
Voyager Instrument Control Panel and Data Explorer 4.0
software (Applied Biosystems, Framingham, USA), respec-
tively. External calibration was performed using a standard
peptide mixture (Calibration Mixture I from Sequazyme Pep-
tide Mass Standard Kit, Applied Biosystems, Framingham,
USA) covering the mass range between 500 and 5000 Da.
The sample solutions were manually spotted on a
stainless steel target plate (100 wells) by the conventional
dried-droplet method. Briefly, the diluted sample solutions
were premixed in a 1 : 1 ratio (v/v) with the matrix solution
Figure 1. The sulphydryl-specific derivatization of
stable-isotope labeling reagent ([d0]- or [d6]-DMMSP) with a
Cys-containing peptide under optimized pH condition.
Figure 2. Structure of the novel stable-isotope labeling
reagent. It consists of three subunits: (1) a reactive leaving
group with specificity towards sulphydryl groups, (2) a
pyrimidine ring to enhance ionization efficiency, and (3) two
methoxyl tags that can incorporate stable isotopes. The
reagent exists in two forms, light (containing no deuteriums)
and heavy (containing six deuteriums).
(
0
6 mg/ml of CHCA in 50% aqueous acetonitrile containing
.1% TFA). Aliquots of 1 µl were deposited on the target and
allowed to air-dry before loading into the mass spectrometer.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 1514–1521
DOI: 10.1002/jms

Pyrimidine-based stable-isotope labeling reagent
1517
to peptides with trypsin. Then the side chains of cysteine
residues are derivatized with [d ]- and [d ]-DMMSP, respec-
0
6
tively. The labeled peptides are combined and directly
analyzed by MALDI-TOF MS for protein identification
and quantification without prior separation. In this pro-
cess, only the peptide containing a cysteine residue can
be labeled by our isotope-labeling reagent, presenting a
mass shift of 138 Da. At the same time, the signal intensi-
ties of labeled Cys-containing peptides are enhanced owing
to the introduction of nitrogenous heterocyclic rings. For
quantitative analysis, the mass difference between the pep-
tides labeled with light and heavy versions of the same
reagent is 6 Da (per cysteine residue) and a series of iso-
topically labeled peptide pairs of labeled peptides can be
identified straightforwardly from the MALDI mass spec-
tra. Relative difference in each protein quantity can then
be determined by measuring the intensity ratio of a given
pair of light/heavy labeled peptide peaks in a MS spec-
trum.
Figure 4. Two-step method for the syntheses of [d0]- and
[
d6]-4,6-dimethoxy-2-(methylsufonyl)pyrimidine.
Synthesis of the labeling reagent
We developed an optimum two-step method based partially
3
6–40
on previous synthetic work.
DMMSP is achieved by treating the commercially available
,6-dichloro-2-(methylthio)pyrimidine with excess sodium
Briefly, the synthesis of
4
metal in methanol, followed by an oxidation with mCPBA in
dichloromethane (Fig. 4). Conveniently, the ‘light’ or ‘heavy’
form of the labeling reagent can be obtained in fairly good
yield using isotopically different solvents (methanol-d
0
or
methanol-d , respectively). All the materials are readily
4
available and inexpensive. The synthetic route is quite
mild and straightforward and does not require any special
techniques.
Reactivity and specificity of [d ]-DMMSP labeling
0
Substrates including various amino acids and a series of
peptides were derivatized with [d ]-DMMSP under different
0
pH conditions, followed by MALDI-TOF mass spectrometric
analysis. The results showed that the reagent’s reactivity
was gradually diminished while decreasing the pH value
of reaction system; that is, the labeling reaction is pH
dependent. Only cysteine or Cys-containing peptide can
react with the labeling reagent under an acidic condition (pH
value within the range of 3.0–6.5) (Table 1). Representatively,
Table 1. The reactivity of [d0]-DMMSP under acidic
conditionsa
Entry
Substrates
Productsb
∗
1
2
–
–
3
4
–
–
–
C
C
K
C (m/z 260)
∗
C (m/z 260)
K (m/z 147)
Y (m/z 182)
HW–6 (m/z 1044)
Y
∗
∗
HW-6 (HCKFWW)
HW-6 (HCKFWW)
DL-10 (DRVYIHPFHL)
ER-14 (EGVNDNEEGFFSAR)
EL-13 (pELYENKPRRPYIL)
HW–6 (m/z 1044)
DL-10 (m/z 1296)
ER-14 (m/z 1570)
EL-14 (m/z 1673)
Figure 3. DMMSP labeling strategy for quantitative analysis of
proteins. Two samples, control and test, are proteolyzed to
peptides with trypsin and then labeled with a ‘light’ (white
triangles) or ‘heavy’ (hatched circles) version of the isotopic
reagent. The labeled peptides are combined and analyzed
directly by MALDI-TOF mass spectrometry for protein
identification and quantification.
a
Similar results were obtained when the pH value ranged from
3.0 to 6.5.
b
Ł
Ł
C
and HW-6 represent the corresponding DMMSP-labeled
amino acid and peptide with a mass increase of 138 Da.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 1514–1521
DOI: 10.1002/jms

1518
J. Zhang et al.
a mass increase of 138 Da was observed from MALDI-TOF
mass spectrum because of the labeling of peptide HW-6
were unlabeled and all detected. This indicated that the
labeling procedure would not complicate the mass spectra
and the following qualitative analyses. For quantification,
one Cys-containing peptide is sufficient, but high coverage
will enhance the reliability of the identification and quan-
tification. Therefore, we developed an optimum procedure
with good coverage, sufficient reactivity, and high specificity
using DMMSP as stable-isotope labeling reagent.
(
Fig. 5). After DMMSP labeling, the signal intensity of Cys-
containing peptide (HW-6) was greatly enhanced compared
to that before labeling. The peaks of other peptides were
all very low, especially that of peptide DL-10 which could
hardly be detected. The spectrum also indicated that the
Cys-containing peptide was specifically and completely
converted into its labeled form in the presence of reducing
agent (DTT).
Quantitative analysis using [d ]- and [d ]-DMMSP
0
6
In order to examine the applicability of the isotope-labeling
reagent for quantitative analysis, two standard solutions
were quantitatively analyzed according to the labeling
strategy described above. The MALDI-TOF mass spectra
for quantitative analyses of cysteine and Cys-containing
peptide (HW-6) are shown in Fig. 6. The combination of
The further optimization of [d ]-DMMSP labeling
and application to protein identification
Considering the complexity of biological systems and the
pH-dependence of labeling reaction, lysozyme was selected
for further investigation. After tryptic digestion, the resultant
0
0
solution was subjected to [d ]-DMMSP labeling and the
‘
light’ and ‘heavy’ labeled samples gave a 6 Da difference
in the molecular weight, which can be visually identified
between m/z 260.2 and 266.3 for cysteine sample in Fig. 6(a),
following MALDI-TOF MS analysis. Finally, the pH value of
the solution was adjusted to 6.5, which promised maximum
reactivity and synchronous sulphydryl-specificity of the
labeling reagent.
Sixteen tryptic fragments of lysozyme including eight
Cys-containing peptides were successfully identified by
MALDI-TOF MS (Table 2). During the procedure, those
Cys-containing peptides were exhausted and completely
converted to their labeled forms while the other peptides
(
and between m/z 1044.4 and 1050.4 for HW-6 sample in
Fig. 6(b)). The intensity ratios of the monoisotopic peaks
existing in pairs were calculated and proven consistent with
the actual sample concentration ratios (1.5). The observed
ratios were 1.54 and 1.57, with the corresponding errors
C
of 2.6% and 4.6%, respectively. The corresponding Na
C
adducts (m/z 1066.4 and 1072.4) and K adducts (m/z 1082.4
and 1088.4) of HW-6 were also observed clearly in pairs.
Their relative intensity ratios provided auxiliary and reliable
information for more accurate quantitative analysis.
Validity of MALDI-TOF MS analysis
While the choice of MS is not critical and either MALDI or
electrospray ionization (ESI) is possible, we chose MALDI
rather than ESI because of its moderate tolerance against
salts, the high speed of analysis, and the comparatively
simple spectra caused by the formation of the singly
charged ions. The liquid chromatography step was also
eliminated in our strategy, avoiding the differential elution
of identical peptides labeled with the H/D isotope pairs in
chromatographic separations.
To examine the overall accuracy and reproducibility of
the MALDI-TOF MS method for relative quantification,
either of the above combined mixtures for quantitative
analysis was premixed with the matrix solution and directly
loaded on the target plate. A total of 200 laser shots were
applied to different locations over the entire surface of each
sample spot. Also, all the mass spectra were cumulated and
averaged to give the final spectrum. This procedure will
enhance the in-spot reproducibility because it minimizes
the influences derived from the inhomogeneous distribution
of analyte within the cocrystallite. The process for loading
the sample was repeated 8 times and the relative intensity
ratios of peaks existing in pairs were recorded. The observed
fluctuations around the theoretical ratios are plotted in Fig. 7.
It is apparent that the samples loaded according to our
procedure exhibited high spot-to-spot reproducibility. The
errors for cysteine and HW-6 were in the range of ꢁ2.6 to 5.3%
and ꢁ2.6 to 4.6%, respectively. The results also demonstrated
that the excess labeling reagent and other impurities such as
Figure 5. The comparison of MALDI-TOF mass spectra of
peptides mixture (Entry 4 in Table 1), (a) before and (b) after
treated with [d0]-DMMSP under pH 4.5.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 1514–1521
DOI: 10.1002/jms

Pyrimidine-based stable-isotope labeling reagent
1519
Table 2. Peptides identified from lysozyme digest and the [d0]-DMMSP-treated mixture
Observed
Matched peptides from
database
m/z
Number
of
cysteines
Number
of added
tags
Before
labeling
After
labeling
Theoretical
No
m/z
Position
Sequence
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
520.2
606.4
874.4
936.4
992.5
1045.6
1276.7
1295.7
1428.7
1434.7
1451.8
1675.9
1753.9
1804.0
2124.1
2678.3
658.1
606.3
874.4
520.3
606.4
874.4
936.4
992.5
1045.5
1276.6
1295.6
1428.7
1434.6
1451.8
1675.8
1753.8
1803.9
2124.0
2678.2
131–134
19–23
33–39
80–86
24–32
135–143
133–143
20–31
52–63
80–91
20–32
116–130
64–79
115–130
33–51
40–63
NRCK
KVFGR
HGLDNYR
WWCNDGR
CELAAAMKR
GTDVQAWIR
CKGTDVQAWIR
VFGRCELAAAMK
FESNFNTQATNR
1
0
0
1
1
0
1
1
0
1
1
0
0
0
1
1
1
0
0
1
1
0
1
1
0
1
1
0
0
0
1
1
1014.4
1030.5
1045.5
1414.7
1433.6
1428.7
1572.8
1589.8
1675.8
1753.9
1803.9
2262.1
2816.4
1
1
1
1
1
1
1
WWCNDGRTPGSR
VFGRCELAAAMKR
IVSDGNGMNAWVAWR
NTDGSTDYGILQINSR
KIVSDGNGMNAWVAWR
HGLDNYRGYSLGNWVCAAK
GYSLGNWVCAAKFESNFNTQATNR
MS method, providing a more rapid method for quantitative
analysis.
The pilot application of DMMSP labeling strategy
to protein samples
As a simple demonstration of this novel labeling reagent’s
ability for effective differential quantification, two lysozyme
tryptic digests with different concentrations were derivatized
0 6
separately with both the [d ]- and [d ] forms of DMMSP, and
combined in the ratio 1 : 1. The final quantification results
by MALDI-TOF MS are summarized in Table 3. An isotopic
pair differing in mass by 6 Da in the spectrum indicated
the presence of a single label. The relative concentration of
protein is directly proportional to its signal intensity. The
errors for lysozyme quantification were all below š4%.
On evaluating the overall DMMSP labeling strategy,
it has several attractive features. First, the selection of
cysteine-containing peptide as the research object permits
the comprehensive identification of proteins, while the
complexity of subsequent analysis is significantly reduced.
Figure 6. Quantitative MALDI-TOF mass spectra of (a) cysteine
and (b) Cys-containing peptide (HW-6) by differentially labeling
with ‘light’ and ‘heavy’ reagents. The theoretical ratios of both
samples are 1.5.
trypsin, urea and additional DTT did not affect the accuracy
of quantification. Therefore, the quantitative analysis can be
achieved at high accuracy and reproducibility using MALDI
Figure 7. The spot-to-spot reproducibility of quantitative
analyses using cysteine and peptide HW-6 as model sample.
Copyright  2007 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2007; 42: 1514–1521
DOI: 10.1002/jms

1520
J. Zhang et al.
Table 3. Comparative quantification of lysozyme digest using [d0]- and [d6]- DMMSP
Observed
m/z
Observed
ratio
Expected
ratio
Error
%
a
Sequenceb
No
∗
1
2
3
4
658.3
1074.4
1414.7
1572.7
NRC K
0.87
0.92
0.88
0.87
0.9
ꢁ3.3
2.2
ꢁ2.2
ꢁ3.3
∗
WWC NDGR
C KGTDVQAWIR
WWC NDGRTPGSR
∗
∗
a
Observed m/z listed here was the corresponding mass of [d0]-DMMSP labeled peptide.
b
∗
C
represented that the cysteine residue was labeled by both [d0]- and [d6]-DMMSP with a mass increase of 138 and 144 Da,
respectively.
Only the peptide containing a cysteine residue presents
a mass shift of 138 Da, leading to easier interpretation
of mass spectra. Also, we could just pay close attention
to the intensity ratios of pairs of labeled Cys-containing
peptides differing by 6 Da in MALDI mass spectra, which
reflect the stoichiometry of the original mixture. Second, the
labeling reagent is less likely to cause overlap with target
peptides in the higher mass range owing to the relatively
small and simple molecular structure of DMMSP. Instead,
the introduction of the pyrimidine ring will increase the
intensities of target peptides in MALDI MS resulting from the
increased basicity of the derivatized cysteine residue, which
facilitates the protein identification. Finally, the procedure is
unaffected by the presence of proteolytic enzymes such as
trypsin or strong denaturants and reductants such as urea
and DTT. There is, therefore, no need for additional steps to
remove such molecules.
Acknowledgements
The authors gratefully acknowledge financial support from the
National Natural Science Foundation of China (20475059, 20632070)
and the Chinese Academy of Sciences (KGCX3-SYW-203-03).
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