RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 683–688

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1842

Determination of sites citrullinated by peptidylarginine

deiminase using 18O stable isotope labeling and mass
spectrometry
Kazuishi Kubota*, Tomoko Yoneyama-Takazawa and Kimihisa Ichikawa
Biomedical Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 29 October 2004; Revised 13 January 2005; Accepted 13 January 2005

Peptidylarginine deiminase (PADI) is an enzyme which catalyzes conversion of arginine residues

into citrulline residues in proteins. Citrullination is known to be related to autoimmune diseases
including rheumatoid arthritis. Previous work in this laboratory succeeded in identifying citrulli-
nated sites of human fibrinogen by mass spectrometry, but discrimination between citrullination
and deamidation of asparagines and glutamine required time-consuming and labor-intensive
inspection of tandem mass spectra. In this work a stable isotope is utilized to improve on a pre-
vious method for the determination of citrullinated sites by mass spectrometry. Since an oxygen
atom is incorporated into the citrulline residue from H2O in citrullination by PADI, peptides citrul-
linated in 50% H18 2 O would show a characteristic isotope distribution different from natural abun-
dance, and thus determination of citrullinated sites is expected to be much easier. To verify the
utility of this new method, the sites of citrullination of human fibrinogen by human PADI4
were investigated using 50% H18 2 O. Compared with the previous method, this new method identi-
fied citrullinated sites more easily and effectively, while both the determined citrullinated sites and
protein sequence coverage were unaltered. Copyright # 2005 John Wiley & Sons, Ltd.

Post-translational modifications regulate various cellular
processes by changing a protein’s activity, localization,
degradation, folding and interaction with other proteins.1,2
Among these modifications, protein deimination converts
protein arginine (Arg) residues into citrulline (Cit) residues,
a process referred to as citrullination.3 Citrullination is cata-
lyzed by a family of Ca2þ-dependent enzymes, peptidylargi-
nine deiminase (PADI; EC 3.5.3.15). So far five PADI isoforms
(PADI1-4 and PADI6) have been cloned in humans, and each
PADI has its own individual characteristics with respect to
expression pattern, substrate specificity, subcellular localiza-
tion, etc.4– 9
PADIs and citrullinated proteins have been reported to be
related to autoimmune diseases,10,11 especially rheumatoid
arthritis (RA).9 Sera from RA patients frequently have
autoantibodies which react with citrullinated proteins, and
these antibodies can be used as a diagnostic marker for
RA.9,12 Moreover, recent large-scale single-nucleotide poly-
morphism (SNP) analysis showed that the PADI4 gene is
associated with susceptibility to RA in Japanese indivi-
duals.13 However, many questions regarding the relation-
ship between RA etiology and PADI remain unanswered.14
To elucidate the involvement of PADIs in RA we
previously identified and compared sites of citrullination of
human fibrinogen by two human PADIs, PADI2 and
PADI4,15 since these PADIs are expressed in RA synovial
*Correspondence to: K. Kubota, Biomedical Research Labora-
tories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku,
Tokyo 140-8710, Japan.
E-mail: kkubot@sankyo.co.jp
tissues,9,16 and citrullinated fibrinogen was detected in these
tissues.17 Detection of citrullinated proteins can be accom-
plished by a colorimetric method,3 by using an anti-modified
citrulline antibody,18 or by amino acid analysis.19 However,
determination of citrullinated sites in a protein needs a more
specific method, such as Edman sequencing.19 Since recent
development of post-translational modification analysis by
mass spectrometry (MS) fulfilled the requirements with
higher sensitivity and specificity,2 we chose to use MS, and
analyzed citrullinated sites of human fibrinogen by liquid
chromatography with tandem mass spectrometry (LC/MS/
MS) after multiple protease digestion.15
Citrullination causes a mass shift of þ1 Da on the Arg
residue (Fig. 1), and thus our previous work searched for
peptides with a þ1 Da shift on the Arg residue in the LC/MS/
MS analysis. Although we succeeded in identifying many
citrullinated sites in human fibrinogen,15 discrimination of
citrullination as opposed to deamidation of asparagine (Asn)
and glutamine (Gln), which leads to the same mass shift of
þ1 Da, was not easy and required careful inspection of the
MS/MS spectra of each candidate peptide. As a result, this
analysis was very time-consuming and labor-intensive.
To overcome these drawbacks and improve throughput, it
was decided to investigate use of a stable isotope labeling
technique for the determination of citrullinated sites in a
protein. The stable isotope labeling technique has been
widely utilized for MS analysis of proteins20–23 and their
post-translational modifications.2,24–26 During citrullination
by PADI, the oxygen atom is incorporated from H2O,9,27 and
thus a peptide or protein citrullinated in H218O should show a

Copyright # 2005 John Wiley & Sons, Ltd.

684 K. Kubota, T. Yoneyama-Takazawa and K. Ichikawa
Figure 1. Characteristic isotope distribution of a peptide citrullinated in 50% H218O. The peptide
incorporates H2O while releasing NH3 during citrullination by PADI. Thus, the peptide citrullinated in H2O
or in H218O shows a mass shift of þ1 or þ3 Da, respectively, relative to the unmodified peptide, in the mass
spectrum. Moreover, the peptide citrullinated in 50% H218O shows a characteristic isotope distribution.
mass shift of þ3 Da on the Arg residue (Fig. 1). Since no
common modification leading to a þ3 Da shift has been
reported, discrimination of citrullinated sites against other
modifications was expected to be easy. Furthermore, if the
protein was citrullinated in 50% H218O, the mass spectrum of
a citrullinated peptide resulting from proteolysis of the
modified protein should show a characteristic isotope
distribution because of the combined spectra of the þ1 and
þ3 Da shifts of the peptide (Fig. 1). Since this artificial isotope
distribution would not exist naturally, citrullinated sites were
expected to be determined much more easily.
The current study validated a method for determination of
citrullinated sites utilizing 50% H218O for stable isotope
labeling. Using human fibrinogen and human PADI4
(hPADI4) as a model experimental system, the new method
was compared with the previous method15 with respect to
three criteria: whether (1) identification of citrullinated sites,
and (2) protein sequence coverage of fibrinogen, would
differ between the two methods, and (3) whether overall
productivity of the method would be improved.
EXPERIMENTAL
Materials
Synthetic peptides corresponding to the human fibrinogen
Aa chain were a generous gift from Dr. K. Sugimoto and T.
Nakata, and purified hPADI4 expressed in E. coli was a
kind gift from M. Nakayama.15 Human fibrinogen was
obtained from American Diagnostics (Pendleton, USA).
Citrullination of peptides
The synthetic peptide (1 mg/mL) was mixed with or
without hPADI4 (20 mg/mL) in 50% H218O containing
100 mM Tris-Cl, pH 7.6, 10 mM CaCl2 and 5 mM dithiothrei-
tol (DTT), and incubated for 2 h at 378C. The citrullination
reaction was stopped by adding EDTA to a final concentra-
tion of 10 mM. The reaction mixture was stored at
208C
and diluted 500-fold with 0.05% formic acid immediately
prior to MS analysis.
Copyright # 2005 John Wiley & Sons, Ltd.
Citrullination of fibrinogen by hPADI4
The human fibrinogen (2 mg/mL) was mixed with or without
hPADI4 in either natural abundance water (H2O) or 50%
H218O containing 80 mM Tris-HCl, pH 7.6, 8 mM CaCl2
and 4 mM DTT, and incubated for 2 h at 378C. The hPADI4
concentration was adjusted to 20 mg/mL for citrullination in
H2O and 160 mg/mL for citrullination in 50% H18 2 O, respec-
tively. The citrullination reaction was stopped by adding
EDTA to a final concentration of 10 mM, and the reaction mix-
ture was stored at
208C until use.
Protease digestion of fibrinogen
The fibrinogen treated with or without hPADI4 in H2O or
50% H218O was digested as described previously.15 Briefly,
the fibrinogen was denatured with 0.1 M NH4HCO3 (pH 7.8)
containing 6 M guanidine-HCl and 10 mM EDTA, and the
denatured fibrinogen was reduced and alkylated with
10 mM DTT and 55 mM iodoacetamide, respectively. The
buffer of the proteins was changed to 20 mM NH4HCO3,
pH 8.0, by gel filtration chromatography (Fast Desalting PC
3.2/10, Amersham Biosciences, Piscataway, USA), and then
the proteins were deglycosylated by peptide:N-glycanase F
(PNGase F, 500 U/mL; New England Biolabs, Beverly,
USA). The deglycosylated proteins were digested separately
for 12 h by three different proteases: trypsin (modified tryp-
sin, Promega, Madison, USA) at 378C; chymotrypsin (Roche
Applied Science, Indianapolis, USA) at 258C; and Glu-C
(Roche Applied Science) at 258C. Adding formic acid stopped
the enzymatic reaction and the samples were stored at
208C
until use.
Mass spectrometry
The mixture of peptides resulting from the digestion was ana-
lyzed by LC/MS/MS. The Q-TOF2 mass spectrometer
(Waters, Milford, USA) was equipped with a CapLC chroma-
tography system (Waters) using a homemade electrospray
ionization (ESI) tip packed with ODS media (Develosil-HG-
3; Nomura Chemicals, Seto, Japan). A sample (5 mL) was
loaded on the column equilibrated with 0.05% formic acid
Rapid Commun. Mass Spectrom. 2005; 19: 683–688

at 1 mL/min; after washing with the same buffer, the flow rate
was then adjusted to 250 nL/min using a homemade splitter.
The peptides were eluted using a linear gradient of 0–30%
acetonitrile over 60 min and ionized by applying 1800 V to
the emitter. The mass spectrometer was operated in the
data-dependent acquisition mode to automatically switch
between MS and MS/MS. Survey mass spectra (m/z 350–
1500) were acquired for 1 s, and the three most intense ions
(doubly, triply or quadruply charged) were isolated and
sequentially fragmented by collision-induced dissociation
by argon gas. To observe characteristic isotope distribution
in the fragment ions, the selection width of the precursor
ions was increased to more than 5 Th units by setting both
the LM resolution and HM resolution to 1.0 (arbitrary units).
Collision energy was set to 14–55 eV depending on the charge
and m/z of the isolated ion. MS/MS spectra (m/z 50–2000)
were acquired for 1 s. The mass spectral resolution at m/z
500 for both the MS and MS/MS measurements was more
than 9000, and m/z error was less than 0.2 Th using external
calibration.
Data analysis
The MS/MS data were searched against the SWISS-PROT
database, specifying each protease used in the sample pre-
paration, using the Mascot program (Matrix Sciences,
London, UK) with consideration of one fixed modification
(carbamoylmethylation of Cys) and several variable modifi-
cations (citrullination of Arg, deamidation of Asn and Gln,
oxidation of Met, and pyroglutamination of N-terminal
Gln). Mass tolerance was set to 0.5 Da for both MS and
MS/MS.
For samples prepared in natural H2O, the criteria for
identification of the citrullinated sites were: (i) appearance of
a peptide of a mass shift of þ1 Da in the sample treated with
hPADI4; (ii) disappearance of the peptide in the sample
without hPADI4; and (iii) reliable assignment of a mass shift
of þ1 Da on the specific Arg residue in the MS/MS spectra of
the peptide by manual inspection.
For samples prepared in 50% H218O, the criteria for the
citrullinated peptides were: (i) appearance of a peptide of a
mass shift of þ1 Da with unusual isotope distribution in the
sample treated with hPADI4; and (ii) disappearance of the
peptide in the sample without hPADI4. When the peptide
had only one Arg residue, the Arg residue was identified as
the citrullinated site. When the peptide had more than two
Arg residues, for identification of the citrullinated site it was
necessary to establish that the characteristic isotope distribu-
tion was caused by a specific Arg residue in the MS/MS
spectra of the peptide.
RESULTS AND DISCUSSION
18
Incorporation of O in the Cit residue
To confirm the incorporation of 18O into the Cit residue, five
10-mer synthetic peptides corresponding to human fibrino-
gen, in each of which one citrullination site had been identi-
fied in a previous study,15 were citrullinated by hPADI4 in
50% H218O and analyzed by LC/MS/MS. As one example,
the case of EGGGVRGPRVV corresponding to human fibri-
nogen Aa chain 11–21 is shown in Fig. 2. As expected, the
Copyright # 2005 John Wiley & Sons, Ltd.
Citrullinated site determination by stable isotope labeling 685
Figure 2. Synthetic peptide citrullinated by human PADI4 in
50% H218O. The synthetic peptide (EGGGVRGPRVV),
corresponding to human fibrinogen Aa chain 11–21, was
treated with or without human PADI4 (hPADI4) in 50% H218O.
Mass spectra of (A) the unmodified peptide treated without
hPADI4 and (B) the monocitrullinated peptide arising from
treatment with hPADI4 in 50% H218O.
citrullinated peptide demonstrates the characteristic isotope
distribution corresponding to 50% incorporation of the 18O
atom in the Cit residue of the sixth Arg residue. On the other
hand, however, we observed a marked decrease in the citrul-
lination rate in 50% H218O compared with H2O, possibly
reflecting an isotope effect.
Therefore, the degree of decrease in the citrullination rate
in 50% H218O compared with in H2O was then examined
using the whole human fibrinogen as the substrate protein.
The citrullination reaction was monitored by the colorimetric
method.28 The decrease of the rate was only slight for the
intact fibrinogen (i.e., the reaction rate appeared almost
unchanged) on adding an 8-fold larger amount of hPADI4 in
50% H218O compared with the corresponding H2O reaction
rate (data not shown). Thus, we used 20 mg/mL of hPADI4 in
H2O and 160 mg/mL in 50% H218O.
Criteria for determination of citrullinated sites
In the previous study,15 where human fibrinogen was citrul-
linated in H2O, discrimination between citrullination on the
Arg residues and deamidation on the Asn or Gln residues
was a difficult problem in practice. Both post-translational
modifications lead to a þ1 Da shift, and deamidation is one
of the most commonly observed modifications which is
known to occur both naturally and artificially.29 In addition
to reports of deamidation of fibrinogen,30 deamidation of
N-glycosylated Asn residues occurs during deglycosylation
by PNGase F. To circumvent false identification of deamida-
tion as citrullination, a control sample without hPADI4 treat-
ment was subjected to analysis in parallel,15 and one of the
criteria for the determination of citrullinated sites was set to
require that the citrullinated peptide in the control sample
(no treatment with hPADI4) was not observed. Furthermore,
to avoid misidentification due to the possibility that citrulli-
nation causes alteration of the cleavage sites of protease
digestion, it was decided15 that reliable assignment of a
Rapid Commun. Mass Spectrom. 2005; 19: 683–688
686 K. Kubota, T. Yoneyama-Takazawa and K. Ichikawa

Figure 3. Citrullinated peptide observed in Glu-C digest of human fibrinogen. Human

fibrinogen was treated with hPADI4 in H2O or 50% H218O, digested by Glu-C, and subjected
to LC/MS/MS analysis. The peptide (GGGVRGPRVVE), corresponding to human
fibrinogen Aa chain 12–22, was observed as a monocitrullinated peptide in 50% H218O
(A) and in H2O (B), and as an unmodified peptide (C), in the mass spectra.

Figure 4. MS/MS spectrum of human fibrinogen citrullinated peptide labeled in 50% H218O.
MS/MS spectrum of the precursor ion observed in Fig. 3(A), the monocitrullinated peptide
corresponding to human fibrinogen Aa chain 12–22. The amino acid sequence and the
assignment of fragment ions are shown. Insets are expanded views around the y6 and y7 ions.
þ1 Da shift on the Arg residue via the MS/MS spectra was the characteristic isotope distribution, the Arg residue should
necessary for identification of citrullinated sites. Since all be identifiable as a citrullinated site without tedious inspec-
Arg, Asn and Gln residues in the peptide are candidates for tion of MS/MS spectra. If there are two or more Arg residues
a þ1 Da shift, careful inspection of MS/MS spectra was in the peptide, the MS/MS spectra must be inspected to
required for this analysis, resulting in the analysis being determine which Arg residue is citrullinated. Considering
very laborious and time-consuming. the small possibility of the incorporation of 18O into the Asn
In the present study using citrullination in 50% H218O, or Gln residue due to deamidation during hPADI4 treatment,
since citrullinated peptides should show the characteristic disappearance of citrullinated peptide in a control sample
isotope distribution (Figs. 1 and 2), clear discrimination without hPADI4 in 50% H218O is required for completely
between citrullination and deamidation would be expected. reliable determination of citrullinated sites as well as in the
Therefore, if there is a single Arg residue in the peptide with natural H2O reaction.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 683–688

Determination of citrullinated sites of fibrinogen
using 18O stable isotope labeling
Fibrinogen is an N-glycosylated protein comprising two sets
of three polypeptide chains termed Aa, Bb and g, that are con-
nected by disulfide bonds.30,31 Some glycosylated proteins
are known to be resistant to proteolytic digestion in solution
without deglycosylation.32 Thus, human fibrinogen was
reduced, alkylated and deglycosylated prior to protease
digestion in this study. Regarding protease digestion, we
used three different enzymes separately to improve protein
coverage, similar to the strategy described previously.33
The LC/MS/MS data obtained were searched separately
against the SWISS-PROT database for assignment, and the
results were combined.
As an example, in the mass spectrum of the peptide
corresponding to human fibrinogen Aa chain 12–22
(GGGVRGPRVVE), the [Mþ2H]2þ ion was observed for the
sample digested by Glu-C (Fig. 3). The characteristic isotope
distribution of the peptide treated with hPADI4 in 50% H218O
indicated that the peptide was monocitrullinated. Unfortu-
nately, this peptide had two Arg residues in the sequence, so
it was not possible to determine the citrullinated site
immediately, and the MS/MS spectrum of the peptide had
to be inspected and interpreted. Even in this case, candidates
for the modification site were just two Arg residues. The y7
ion in the MS/MS spectrum of the peptide (Fig. 4) showed the
characteristic isotope distribution of 18O labeling, but the y6
ion did not, which clearly demonstrated citrullination of the
fifth Arg residue in the peptide.
By closer inspection of the MS spectra of the peptides from
human fibrinogen, we observed that the second isotope signal,
corresponding to the incorporated 18O, was generally smaller
than that theoretically predicted. This phenomenon was not
observed for the synthetic peptides, as is clearly illustrated by
comparing Fig. 3 (fibrinogen) with Fig. 2 (synthetic peptide).
This might be due to a back-exchange reaction catalyzed by
hPADI4 after citrullination and before protease digestion.
Removal of the Ca2þ ion causes citrullination by hPADI4 to
stop, but the back-exchange reaction might not.
Figure 5. Citrullinated sites identified in human fibrinogen Aa chain. The amino acid sequence of the human
fibrinogen Aa chain is shown. Identified citrullinated sites, indicated by circles, were in complete agreement
between analysis citrullinated in 50% H218O and in natural H2O. Underlined sequences were covered by MS/
MS spectra in 50% H218O, and dashed-lined sequences were covered by MS/MS spectra in H2O.
Copyright # 2005 John Wiley & Sons, Ltd.
Citrullinated site determination by stable isotope labeling 687
Table 1. Determined citrullinated sites of fibrinogen
Coverage (%)a
Determined
Fibrinogen H2O 50% H218O citrullinated sites
Aa chain 75% 69% R16, R23, R252,
R406, R407,
R554, R572
Bb chain 89% 92% R14, R23
g chain 75% 73% R5
a
Percentage of amino acid sequence covered by MS/MS spectra.
However, even considering this signal intensity reduction
of the second isotope signal, the isotope distribution
characteristics remain sufficiently striking (Fig. 3) that we
could easily identify the citrullinated peptides from only
their mass spectra. Moreover, there were few cases that
needed inspection of MS/MS spectra because of multiple Arg
residues, and, even in such cases, determination of citrulli-
nated sites was much easier than described above (Fig. 4).
Taken together, we could accomplish more high-throughput
determination by the 18O labeling method than by the
previous method.
One of our criteria for the 18O labeling method was
disappearance of the citrullinated peptide in the control
sample without hPADI4. Since in the present work all
peptides labeled with 18O in the hPADI4 sample were not
observed in the control sample without hPADI4, it may be
possible in general to omit analysis of a control sample. If this
turns out to be the case, this 18O labeling method may become
an even higher-throughput approach.
Finally, the citrullinated sites determined by this new 18O
labeling method were completely identical with those found
using our previous method,15 even though the amount of
hPADI4 used for each citrullination reaction was different.
Table 1 summarizes the comparison of the 18O labeling
method with the previous method, and Fig. 5 illustrates the
identified citrullinated sites and protein sequence coverage of
the human fibrinogen Aa chain as an example. The protein
sequences covered by the MS/MS spectra were almost
Rapid Commun. Mass Spectrom. 2005; 19: 683–688
688 K. Kubota, T. Yoneyama-Takazawa and K. Ichikawa
equivalent between these two methods (Table 1), although one
part of the sequence was covered in only one method (Fig. 5).
This was attributed to a limitation of the data collection rate in
acquiring MS/MS spectra. Although use of multiple digestions
in our methods decreases the probability of overlooking
citrullinated sites, it was gratifying to obtain complete
agreement of citrullinated sites between the two methods.
Additional repeated LC/MS/MS experiments would be useful
to avoid false negatives. Moreover, automatic selection of
peptide ions with an unusual isotope distribution in the mass
spectra will help to avoid overlooking candidate peptides for
MS/MS analysis, and would increase the chance of detecting
low-abundance citrullinated sites.
CONCLUSIONS
We have developed a new method to determine citrullinated
sites using 18O labeling. This method demonstrated (1) com-
plete agreement of identified citrullinated sites, (2) equivalent
sequence coverage with MS/MS spectra, and simultaneously
(3) higher-throughput determination, compared with a pre-
vious method.15 Rapid determination of citrullinated sites
would be useful for characterization of PADIs, would promote
elucidation of the role of citrullination and PADIs in the physio-
logical context, and eventually may contribute to the develop-
ment of PADI inhibitors as drugs.
Acknowledgements
We especially thank Ms. M. Nakayama-Hamada for provid-
ing the purified hPADI4 and experimental suggestions, and
critical reading of the manuscript. We also acknowledge
Dr. K. Sugimoto and Ms. T. Nakata for providing the syn-
thetic peptide, and Ms. M. Kubota for encouragement and
helpful discussion.
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