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Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with monopotassium phosphoramidate. The results were confirmed by 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results indicate that the ECD-MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.
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Biosci. Rep. (2010) / 30 / 433–443 (Printed in Great Britain) / doi 10.1042/BSR20090167
Electron capture dissociation mass spectrometric
analysis of lysine-phosphorylated peptides
Karolina KOWALEWSKA*, Piotr STEFANOWICZ*, Tomasz RUMAN†, Tomasz FR ˛ACZYK‡, Wojciech RODE‡ and
Zbigniew SZEWCZUK*1
*Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie Street, 50-383 Wrocław, Poland, †Department of Biochemistry and
Biotechnology, Faculty of Chemistry, Rzesz´
ow University of Technology, 6 Powsta´
nc´
ow Warszawy Ave. 35-959 Rzesz´
ow, Poland, and
‡ Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland
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Synopsis
Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although N-
phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in
terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture
dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of
three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with
monopotassium phosphoramidate. The results were confirmed by 1H NMR and 31P NMR studies. The ECD method
was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main
advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results
indicate that the ECD–MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities
of the obtained lysine-phosphorylated peptides under various conditions were also tested.
Key words: electron capture dissociation (ECD), phosphorylation, phospholysine, post-translational modification
INTRODUCTION
Protein phosphorylation belongs to the most important post-
translational modifications, is involved in many signal trans-
duction pathways and plays a significant role in mechan-
isms responsible for the regulation of cellular functions. The
O-phosphorylation of serine, threonine and tyrosine residues
has been extensively studied, since the high stability of O-
phosphorylated products has permitted the analysis of this
modification with many different techniques [1], especially MS
[2,3].
However, other amino acid residues also undergo phosphoryla-
tion, among them the basic amino acids histidine, lysine and
arginine. With the latter amino acid residues, the modification
results in the formation of a phosphoramidate bond (P–N bond),
which is highly unstable under acidic conditions and easily under-
goes hydrolysis. Therefore, N-phosphorylated proteins are diffi-
cult to analyse, and detection of N-phosphorylated peptides using
MS creates problems due to low abundance [4] or spontaneous
gas-phase dephosphorylation [5].
............................................................................................................................................................................................................................................................................................................
Abbreviations used: CID, collision-induced dissociation; ECD, electron capture dissociation; ESI–MS, electrospray ionization MS; ETD, electron transfer dissociation; HMBC,
heteronuclear multiple bond correlation; PKG, protein kinase G.
1To whom correspondence should be addressed (email szewczuk@wchuwr.pl).
Protein N-phosphorylation, compared with O-phosphory-
lation, is still poorly explored in terms of products and mech-
anisms. A considerable interest has been focused on protein
histidine phosphorylation, recognized as an important modific-
ation in prokaryotes and eukaryotes, specific histidine kinases
being involved in signalling systems called the two-component
regulatory systems [6–8]. Histidine was also the first synthetic-
ally N-phosphorylated basic amino acid whose chemical prop-
erties have been well studied [9]. The histidine modification, in
spite of susceptibility to acid hydrolysis, could be analysed us-
ing MS both in peptides and proteins, using different analytical
techniques such as LC–MS (liquid chromatography MS), ESI–
MS (electrospray ionization MS) and MALDI–TOF-MS (matrix-
assisted laser-desorption ionization–time-of-flight MS), and dif-
ferent fragmentation methods, mainly CID (collision-induced
dissociation), ETD (electron transfer dissociation), EDD (elec-
tron detachment dissociation) and ECD (electron capture disso-
ciation) [10–13].
Protein N-phosphorylation may also occur on arginine and
lysine residues (reviewed recently by Besant et al. [14]). The
presence of protein arginine and lysine kinases in cells has
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K. Kowalewska and others
been reported [15]. While several reports concerned arginine
kinases involved in cellular metabolic pathways [16,17], N-
phosphorylation of the lysine side chain has been much less in-
vestigated. Specific protein lysine kinases and the corresponding
phosphatases (phosphoramidases) were found in eukaryotic cells
[14], e.g. rat liver nuclear protein arginine kinase with an addi-
tional lysine kinase activity [18], protein lysine kinases involved
in histone H1 phosphorylation [19] and bovine liver protein lysine
phosphatases [20]. The involvement of lysine phosphorylation in
cellular signalling or metabolic pathways, albeit possible, has not
been shown yet.
Although lysine phosphorylation is an important protein modi-
fication, it is difficult to detect by ESI–MS. The main reason is
the low abundance of the phosphopeptide ions as compared with
their non-modified counterparts. Usually, formic acid or acetic
acid is added to aid protonation of the analyte molecules during
electrospray. Therefore, relatively rapid hydrolysis of the phos-
phoramidate group under acidic conditions may also complicate
the application of ESI–MS to the analysis of phospholysine-
containing peptides.
MS has become an alternative to the more traditional meth-
ods, e.g. NMR, two-dimensional gel electrophoresis, HPLC ana-
lysis and proteolytic digestion using Edman degradation of 32P-
labelled peptides, for analysis of phosphorylated peptides [21].
The ESI–MS method may be particularly useful to evaluate the
extent of phosphorylation in a protein molecule. In contrast with
other analytical methods, MS experiments can be performed suc-
cessfully even on sub-picomolar amounts of a mixture of pro-
teins, as long as the analytes possess different molecular mosses.
Moreover, MS fragmentation techniques may be used to charac-
terize the modification sites with the amino acid residue resolu-
tion.
Although the most common method of fragmentation, CID,
is very useful for the sequencing of O-phosphorylated peptides,
its application to N-phosphorylation is limited as a consequence
of considerable losses of the phosphate group HPO3from the
phosphoramidate bond even at relatively low collision energy
conditions. Therefore, the resulting CID spectra are dominated
by the dephosphorylated forms of the fragments.
ECD performed on Fourier transform mass spectrometers
was shown to be useful for the characterization of labile post-
translational protein modification [22], including non-enzymatic
modifications of basic side chains [11,23]. No fragmentation
of phosphorylated histidine residues was observed during ECD
experiments [24,25]. This suggests that ECD may be a useful
method for analysis of peptides containing the N-phosphorylated
lysine side chain, although such an analysis has not been repor-
ted to date. Recently, we used the ECD method for analysis
of distribution of deuterium along the sequence of a protein
molecule undergoing the hydrogen exchange under conditions
of a high-pressure denaturation [26]. Although fragmentation of
deuterium-labelled compounds using the CID method is known to
indicate the migration of deuterons (hydrogen scrambling) [27],
which makes the analysis impractical, we found that the ECD
fragmentation allows for ambiguous recognition of deuterated
peptide bonds in a protein molecule. The current study presents
an analysis using ESI–MS of the lysine phosphorylation products
obtained by the reaction of peptides with monopotassium phos-
phoramidate. Stabilities of peptides containing N-phosphorylated
lysine in solution were also analysed. The comparison between
the two MS fragmentation types, CID and ECD, pointed to ECD
as a better method for localization of N-phosphorylation on the
lysine residue. In addition, the applicability of ECD–MS was
tested to study the regioselectivity of peptide N-phosphorylation.
MATERIALS AND METHODS
Materials
Peptides
Three peptides, known to be the substrates or inhibitors of specific
protein kinases (Table 1), were chosen for the investigation. Each
peptide contained one or two lysine residues localized at different
positions in the sequence. All analysed peptides were purchased
from Sigma–Aldrich.
Peptide N-phosphorylation
Peptide N-phosphorylation was performed using a protocol previ-
ously described by Wei and Matthews [28]. The phosphorylation
agent, monopotassium phosphoramidate, was prepared by the
classical Stokes’ method [29]. In the standard procedure, pep-
tide and monopotassium phosphoramidate were used at a ratio of
1:40 (w/w). The peptide (0.5 mg) sample was dissolved in 10 mM
ammonium bicarbonate and the pH of the solution was adjusted
to 8 with 0.1 M NaOH. After the addition of 20 mg of mono-
potassium phosphoramidate, the mixture sample was stirred for
24 h at room temperature (22C) (Figure 1).
Sample preparation
For the MS experiments, the phosphorylated sample was desalted
using Sep-Pak Plus C18 Cartridges (Waters Corporation). The
column was prepared by washing alternately with acetonitrile
(POCH, Poland, HPLC grade) and deionized water. The sample
was loaded directly on a Sep-Pak column and washed five times
with small portions of the deionized water. The phosphorylated
peptides were eluted with 60 % aqueous solution of acetonitrile
(1 ml).
Methods
ESI–MS
All MS experiments were performed on an Apex-Qe Ultra
7T instrument (Bruker Daltonics, Bremen, Germany) equipped
with a dual ESI source and a heated hollow cathode dispenser.
The instrument was operated in the positive-ion mode and cal-
ibrated with the TunemixTM mixture (Bruker Daltonics). The
mass accuracy was better than 5 ppm. Analysis of the ob-
tained mass spectra was carried out using a Biotools (Bruker
Daltonics) software. The instrumental parameters were as
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2010 Biochemical Society
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which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Detection of phospholysine in peptides
Table 1 Model peptides used in experiments and their biological activity
Peptide sequence Biological activity References
RKRSRAE Selective substrate of PKG (protein
kinase G) with a strong
preference for PKG Iαover PKG II
[30]
RKRARKE An inhibitor of the cGMP (cyclic
guanosine monophosphate)-
dependent protein kinase
[31]
PLSRTLSVAAKK A part of a glycogen synthase
sequence; an effective substrate
of CaM (calmodulin) kinase II and
PKC (protein kinase C)
[32,33]
Figure 1 Scheme of the peptide phosphorylation on the ε-amino group of lysine
rt, room temperature.
follows: scan range, 100–1600 m/z; dry gas, nitrogen; temper-
ature, 200C; potential between the spray needle and the orifice,
4.2 kV.
CID
The precursor ions were selected on the quadrupole and sub-
sequently fragmented in the hexapole collision cell. Argon was
used as a collision gas. The obtained fragments were directed
to the ICR mass analyser and registered as an MS/MS (tandem
MS) spectrum. The collision energy of 20 V was applied in the
hexapole collision cell.
ECD
The mass spectrometer was equipped with a heated hollow cath-
ode dispenser, which was operated at 1.7 A for the ECD exper-
iments. The precursor ions were selected on the quadrupole and
directed to the ICR cell where they were fragmented. The para-
meters were set as 150 ms for the ECD pulse length, and ECD
bias was 0.8 V.
NMR analysis
All NMR spectra were obtained with a Bruker Avance spectro-
meter operating in the quadrature mode at 500.13 MHz for 1H
and 202.46 MHz for 31P nuclei. The residual peaks of deuter-
ated solvents were used as internal standards in the 1HNMR
method. 31P NMR spectra were recorded at 277 K, both with
and without proton decoupling. The internal standard used in
31P NMR was inorganic phosphate (Pi), showing resonance at
2.15 ppm (pH 7.8), 2.05 ppm (pH 7.5), 1.65 ppm (pH 5.0) and
0.0 ppm (pH 1.5). Additionally, chemical shifts were checked
with external reference (85 % H3PO4). All samples were ana-
lysed using the gradient-enhanced 1H-31P HMBC (heteronuclear
multiple bond correlation) method, with the HMBC experiments
optimized for long-range couplings using different 3JPHvalues
(1–20 Hz). The 1H NMR spectra were obtained with and without
the use of the HDO suppression method. All buffer solutions used
for NMR spectroscopy were based on deuterium oxide of 100%
2H purity (Armar Chemicals AG).
RESULTS AND DISCUSSION
Three model peptides were phosphorylated with monopotassium
phosphoramidate and analysed using MS, the ESI–MS spectra
confirming that the reaction yielded N-phosphorylated products.
The peptides are known substrates or inhibitors of protein kinases
(Table 1). All lysine-phosphorylated peptides were stable in 60%
aqueous acetonitrile solution (pH 7) at room temperature (22C).
No degradation product was observed in the ESI–MS spectra
if the samples were incubated at 4C for 1 week. On the other
hand, the phosphorylated peptides were unstable under acidic
conditions. At pH 2–3 (in 10% formic acid), the half-time for
phosphopeptide at room temperature was approx. 20 min. Thus,
the lysine-phosphorylated peptide stability was similar to that
described previously for phosphohistidine [12].
The possibility of arginine side-chain phosphorylation under
the conditions employed was excluded by various NMR and
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K. Kowalewska and others
ESI–MS experiments (not described in the Materials and meth-
ods section). We performed the NMR-controlled pH-dependent
phosphorylation experiments under conditions similar to those
described in the present paper. Our results clearly showed that the
31P NMR spectrum of arginine-phosphoramidate post-reaction
mixture contains no phosphoarginine resonance (singlet reson-
ance) over a pH range from 4.0 to 9.0. The traces of phosphoar-
ginine product were found only for reaction mixtures incubated
at pH 10 and above (we analysed the pH range 3–12), whereas ex-
periments conducted for lysine proved that phospholysine (triplet
resonance) reacts with phosphoramidate ions over the pH range
5–12. A similar conclusion was also obtained on the basis of our
ESI–MS experiments. Therefore, the chemical phosphorylation
employing monopotassium phosphoramidate in pH 8 takes place
exclusively at amino groups, even if arginine residues are present
in the peptide chain.
The ESI–MS spectra (Figure 2a) of the phosphorylated
RKPRSRAE peptide (where KPis the phosphorylated lysine
residue), recorded in the positive-ion mode, present the main
peak at m/z 491.755 corresponding to the monophosphorylated
compound ([MP+2H]2+). In these spectra, the abundant peak
of the phosphorylated peptide is accompanied by its unphos-
phorylated form. There are additional peaks at m/z: 502.746,
510.734, 513.738 and 521.725 that correspond to 2+ions of
the phosphopeptide containing metal cations: [MP+H+Na]2+,
[MP+H+K]2+,[M
P+2Na]2+and [MP+K+Na]2+respect-
ively. Interestingly, no metal adducts were apparent in the unphos-
phorylated peptide spectrum. This may suggest that the presence
of the phosphoramidate group increases the affinity of the peptide
towards the metal ions. The latter effect is observed also in the
ESI–MS spectra of the other analysed model peptides.
The most abundant peak at 491.7 m/z, corresponding to the
[MP+2H]2+ion, was chosen as a precursor for the fragmenta-
tion both by CID and ECD. Figure 2(b) shows the CID spec-
trum of the products obtained by phosphorylation of RKRSRAE
peptide. The number of identified fragments is not sufficient
to establish the phosphorylation site. This may be due to the
extensive neutral losses of HPO3(80 Da) and water molecule
(18 Da). The most abundant peak corresponds to the loss of 98
Da. Although phosphoramidates cannot directly eliminate H3PO4
molecules because of merely three oxygen atoms being available
in the amidate, a concerted loss of one H3PO4molecule from
N-phosphorylated peptides cannot be excluded. Recently, Klein-
nijenhuis et al. [12] observed also that collisional activation of
the histidine-phosphorylated peptide results in the extensive loss
of H3PO4. The authors proved the origin of the lost water to be
an aspartic acid residue adjacent to the phosphorylated histidine
residue. Therefore, although the phosphorylated lysine residue in
RKPRSRAE is surrounded by arginine residues, the eliminated
water molecule may derive from the Ser4or Glu7residues.
The spectrum in Figure 2(b) is dominated by ions correspond-
ing to the elimination of HPO3and the water molecule, whereas
the abundances of the backbone fragments are relatively low,
with many unidentified fragments observed. The CID spectrum
is difficult to analyse and the localization of the peptide phos-
phorylation site is complicated.
A different fragmentation pattern was observed in the spec-
trum obtained following the ECD fragmentation of RKPRSRAE
(Figure 2c). In contrast with CID, the ECD fragmentation of the
[MP+2H]2+ion retains the phosphorylation of the majority of
fragments containing Lys2. On the other hand, in the obtained
cn-andz
n
-series of ions, phosphorylated and unphosphorylated
species are observed, indicating that even the ECD fragmenta-
tion causes certain level of dephosphorylation. In spite of a higher
abundance of the fragments containing phospholysine and dom-
ination of the corresponding peaks in the spectrum, a partial de-
phosphorylation and the lack of certain ions make the spectrum
interpretation difficult. The ECD fragmentation pattern suggests
that the phosphoramidate group is located mainly on the lysine
side chain, although a partial phosphorylation of the N-terminal
amino group cannot be excluded.
The analysed peptide of the RKRARKE sequence encom-
passes two lysine residues – Lys2and Lys6. Although the pep-
tide may be expected to yield a doubly phosphorylated product,
following the reaction with an excess of the phosphorylating
agent, the ESI–MS spectrum (presented in Figure 3a) shows that
the predominant peak corresponds to a singly phosphorylated
product. To simplify interpretation of the spectrum, the ESI–
MS analysis was performed using 2% formic acid as a solvent
preventing metal ion co-ordination to the phosphopeptide, ob-
served at higher pH values. Although the phosphoramidate bond
is known to hydrolyse quickly under acidic conditions, we found
the phosphopeptides to be stable for long enough to perform the
MS experiment (t1/230 min at room temperature).
The mass spectrum (Figure 3a) does not show any peaks com-
ing from the metal-ion adducts. The main peaks result from the
ionization of the unphosphorylated [m/z 472.303 (+2)], mono-
phosphorylated [m/z 512.287 (+2)] and diphosphorylated [m/z
552.270 (+2)] peptide. The unphosphorylated peptide ion dom-
inates (m/z 315.20) in its triply protonated form. The intensity
of the diphosphorylated peptide ions [represented by m/z 552.27
(+2) and 369.24 (+3)] is low. The latter phenomenon may be
a result of either the phosphorylation procedure used being cap-
able of modifying only one lysine residue in RKRARKE or the
neutralization of the molecule’s overall charge by the phosphory-
lation, which makes the ionization more difficult.
The ECD experiment allowed us to localize the phosphoryla-
tion site in doubly and triply charged precursor ions (Figures 3b
and 3c respectively) of the monophosphopeptide. The ECD ana-
lysis performed on the doubly charged ion ([MP+2H]2+, Fig-
ure 3b) is represented predominantly by three series of fragment
ions: a longer series of singly phosphorylated ions zn
PcnP,and
shorter series of non-phosphorylated cnand znions. The mass
peaks, corresponding to the species containing the phosphor-
amidate group, are characterized by a higher abundance.
The ECD fragmentation, performed on the triply charged,
singly phosphorylated peptide ion ([MP+3H]3+) (Figure 3c),
resulted in a better sequence coverage. The cn-andz
n
-series
are almost complete and the number of identified phosphorylated
fragment ions is sufficient to sequence the peptide and to local-
ize the phosphorylation site. The presence of a series of peaks
in the spectrum, corresponding to z2
P,z
3
P,z
4
Pand z5
P,
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2010 Biochemical Society
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Detection of phospholysine in peptides
Figure 2 ESI–MS spectra of the phosphorylated RKPRSRAE peptide
(a) ESI–MS spectrum recorded in the positive-ion mode, (b) CID fragmentation spectrum (precursor ion m/z 491.7) and
(c) ECD fragmentation spectrum (precursor ion m/z 491.7). K* represents possible phosphorylation on the lysine ε-amino
group. The fragmentation pattern is shown in each spectrum. cn- and z
n-fragmentation ions with the index P present the
phosphorylated fragment of the peptide.
suggests that the phosphorylation site is located on Lys6, whereas
the presence of peaks corresponding to c3P,c
4Pand c5P, indicates
that the phosphoramidate may be also present on Lys2. Thus,
the ECD fragmentation spectrum indicates both lysine residues
to undergo N-phosphorylation. The peptide’s chemical modific-
ation does not appear regioselective, albeit the phosphorylation
levels of Lys2and Lys6are different and the relative intensity of
fragment ions suggests the modification of Lys6to be preferred.
The third model peptide, PLSRTLSVAAKK, contains a se-
quence of two lysine residues located at the C-terminus (Lys11
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K. Kowalewska and others
Figure 3 ESI–MS spectrum of RKPRARKPE
(a) Mass spectrum of the phosphorylation products of RKRARKE, (b) ECD fragmentation spectrum (precur sor ion m/z
512.3, [MP+2H]2+) and (c) ECD fragmentation spectrum (precursor ion m/z 341.9, [MP+3H]3+). K* represents a possible
phosphorylated lysine residue.
and Lys12). The positions of lysine moieties might be expected
to influence the N-phosphorylation regioselectivity. The ESI–MS
spectrum presents the peak masses derived from the monophos-
phorylated PLSRTLSVAAKK peptide, with the measurement
done in 2 % formic acid to aid the peptide protonation. Because
of a relatively rapid hydrolysis of the phosphoramidate, the mass
experiment was performed without delay just after the addition
of the acid.
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Detection of phospholysine in peptides
In the ESI–MS spectrum (Figure 4a), only peaks correspond-
ing to the singly phosphorylated peptide are observed, with the
abundance of non-phosphorylated and diphosphorylated peaks
being low. The main peak in the spectrum at m/z 675.883
represents a doubly charged, singly phosphorylated peptide
[MP+2H]2+. Two other forms, singly and triply charged, are
apparent at m/z 1350.754 and 450.923 respectively. It is of note
that the phosphorylated peptide is relatively resistant towards
hydrolysis and does not lose the phosphoramidate group in 2 %
formic acid even after 20 min.
To check the resistance of the modification in phosphorylated
PLSRTLSVAAKK, the sample was incubated with 10 % formic
acid. The ESI–MS spectra, recorded after 2, 10 and 20 min (Fig-
ure 4b), show evolution with time pointing to an apparent slow de-
phosphorylation of the peptides. While the singly phosphorylated
form is still predominant after 2 min, the following 20 minincub-
ation period results in an increase in the peaks corresponding to
the unphosphorylated species. We also examined the stability of
the phosphorylated lysine residues in the phosphopeptide solu-
tion (60 % aqueous acetonitrile) stored at 20C. Surprisingly,
the ESI–MS spectrum, recorded for the lysine-phosphorylated
peptides tested, remains unchanged even after a 6-month incuba-
tion period (Figure 4c), indicating that phospholysine containing
peptides can be stored at low temperature and neutral pH.
To check whether the peptide was N-phosphorylated regio-
selectively, fragmentation was performed using the CID and ECD
methods (Figures 5a and 5b). The doubly charged, singly phos-
phorylated ion (MP+2H)2+of the PLSRTLSVAAKK peptide
was chosen (m/z 675.883) for the fragmentations. The CID spec-
trum shows, beside a majority of all ions derived from the non-
phosphorylated form, only three peaks corresponding to the
phosphorylated peptide fragments and several unidentified peaks.
Although the number of fragment ions is sufficient for the peptide
sequencing, it is difficult to establish the phosphorylation site. The
ECD spectrum provides much more information. The resulting
cn-andz
n
-fragment ions cover 100 % of the peptide sequence.
Furthermore, there are four identified fragment ions, bearing the
phosphoramidate moiety. The intensity of certain ions belonging
to the zn
Pseries was very low and, therefore, the correspond-
ing peaks were not pointed in Figure 5(b). The presence of the
phosphorylated c11Pion proves that Lys11 is phosphorylated, at
least partially. On the other hand, the occurrence of the z1
Pphos-
phorylated ion can be explained only assuming a partial phos-
phorylation of Lys12. Thus, the analysis of the ECD spectrum
reveals that both Lys11 and Lys12 moieties are phosphorylated
to some extent. Fragmentation of the PLSRTLSVAAKK pep-
tide phosphorylation products, performed by the ECD method,
results in a better sequence coverage and significantly higher re-
tention of the phosphoramidate group, as compared with the CID
method. On the other hand, the fragmentation behaviour of the
lysine-phosphorylated peptide reflects a high lability of the phos-
phoramidate moieties. Even with the ECD technique, significant
phosphate-related losses are observed. This observation correl-
ates well with the recently reported electron-based dissociation
(ECD and ETD) of phosphorylated histidine in polypeptides [11].
Our results demonstrate that, although in the process of electron-
based dissociation the phosphorylated lysine residue shows a
lability similar to that of the phosphorylated histidine residue,
it may be sequenced using ECD. On the other hand, the de-
phosphorylation level observed during the ECD fragmentation is
surprisingly high. The electron-based fragmentation methods are
believed to be extremely selective with respect to peptide bonds
[34], and consequently, the ECD should not influence the modi-
fications of peptides. Our results suggest a relatively low stability
of the phosphoramidate bond in the gas phase, compared with
other known post-translational modifications.
NMR analysis was performed on unphosphorylated and phos-
phorylated peptides, RKRARKE and PLSRTLSVAAKK, to con-
firm correctness of the ECD–MS analysis. The 1H NMR chem-
ical shift of the RKRARKE lysine ε-CH2moiety found in
the non-phosphorylated peptide spectrum is approx. 2.93 ppm.
The 1H NMR spectrum of the phosphorylated RKRARKE
peptide, compared with non-phosphorylated RKRARKE pep-
tide, shows clearly a new multiplet resonance at 3.08 ppm,
strongly correlated with that of ε-CH2of the phosphorylated
lysine moiety. The observed downfield shift of the ε-CH2moi-
ety of the phosphorylated form (0.15–0.25 ppm) is typical of
phosphorylated/non-phosphorylated aliphatic amino acid side-
chain systems [35,36]. The 31P NMR spectrum of the phos-
phorylated peptide shows a resonance at approx. 7.0 ppm.
The 1H NMR spectrum of phosphorylated peptide PLSRTLS-
VAAKK contains a multiplet resonance at 3.06 ppm, correspond-
ing to the phospholysine ε-CH2moiety, and the resonances of the
non-phosphorylated lysine ε-CH2moiety visible at 2.90 ppm,
with relative integrals of 1:1 respectively. The 31P NMR spec-
trum of the phosphorylated peptide clearly shows a new triplet
resonance at 7.6 ppm (3JPH=6.8 Hz, Figure 6), with the coup-
ling constants in the range typical of the three-bond P–H
couplings [37,38]. It is of note that the latter resonance appears
to correspond to a single form of phospholysine, as with proton-
decoupling applied. The 31P NMR spectrum shows a single, nar-
row (half-width 2.67 Hz), symmetrical singlet resonance at 7.6
ppm (Figure 6). The previously mentioned triplet form of the
resonance in the 31P NMR spectrum results apparently from the
heteronuclear coupling of phosphate phosphorus with two mag-
netically equivalent hydrogen atoms of the ε-CH2methylene
group of the N-phosphorylated lysine moiety of the PLSRTLS-
VAAKK peptide.
The gradient-enhanced 1H-31P HMBC (see Figure 6c) proved
that the above-mentioned P-coupled methylene hydrogen atoms
show 1H NMR shifts of approx. 3.1 ppm, in agreement with
the 1H NMR spectrum of the phosphorylated PLSRTLSVAAKK
peptide. In view of the analysis of COSY and 1H-13CHSQC
(heteronuclear single-quantum coherence) spectra of the phos-
phorylated peptide, as well as the data on several homopeptide
derivatives [39], the α-CH proton of the lysine moiety with a
free carboxylic group (Lys12,δαCH =3.74 ppm) should not have
the phosphate group at the side chain. Consequently, the other
lysine moiety (Lys11,δαCH =3.80 ppm) must have the phos-
phoramidate group in the ε-CH2region. In view of the analysis
of the NMR data, the presence of the O-phosphoserine moi-
ety in the analysed peptides is not apparent. The three-bond
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K. Kowalewska and others
Figure 4 ESI–MS spectra of the products of phosphorylation of the PLSRTLSVAAKK peptide
(a) ESI–MS spectrum in positive-ion mode in 2 % formic acid. (b) ESI–MS spectra in 10 % formic acid after : A, 2 min; B,
10 min; and C, 20 min. (c) ESI–MS spectrum of the phosphorylated peptide sample after 6 months storage at 20C.
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440 C
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2010 Biochemical Society
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which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Detection of phospholysine in peptides
Figure 5 Fragmentation spectra of the phosphorylated PLSRTLSVAAKK peptide
(a) CID fragmentation spectrum (precursor ion m/z 675.9; [MP+2H]2+) and (b) ECD fragmentation spectrum (precursor
ion m/z 675.9; [MP+2H]2+). K* represents a possible phosphorylated lysine residue.
Figure 6 31P NMR spectrum (A), 31 P NMR spectrum with proton decoupling (B) and 1H-31P HMBC spectrum (C) of the
PLSRTLSVAAKK peptide KPmoiety
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K. Kowalewska and others
coupling between serine CH2and phosphorus would result in
a two-doublets resonance pattern. Moreover, the 1H-31PHMBC
spectra indicate the phosphoserine-, and also thiophosphoserine-
CH2, group to show 1H chemical shifts in the 3.6–4.2 ppm range
[35,36,40]. Furthermore, the observed resonances do not belong
to the phosphorylated arginine or glutamate moieties, as would be
expected with those singlet resonances in 31P NMR. Finally, the
triplet resonances could not originate from the phosphorylated
N-terminal end of a peptide, as a doublet 31P NMR resonance
would have to result from the proximity of the α-CH moiety.
It is of note that, while in view of the NMR results the reaction
appears rather regiospecific, the ECD spectra suggest modific-
ation of both Lys11 and Lys12. In our opinion, the discrepancy
results from different characteristics of the two methods, with
the former providing quantitative results, but not always able to
detect minor constituents, and the latter allowing a higher sensit-
ivity, but not necessarily a quantitative evaluation.
CONCLUSIONS
Comparative MS analyses are presented, accompanied by
1HNMRand31P NMR studies, of model peptides non-
phosphorylated and N-phosphorylated on lysine residue(s). Al-
though the modified peptides, monitored by MS, proved labile
under acidic conditions in accordance with the well-known phos-
phoramidate acid-lability, neutral solutions of the peptides could
be stored for a long time at 20C without losing the phosphor-
amide group.
Two fragmentation methods, ECD and CID, were compared
as tools for the analysis of the N-phosphorylation products. The
results pointed to the ECD fragmentation as an advantageous
method, allowing a significant reduction of the neutral losses
related to the phosphoramidate moiety and permitting, in most
cases, the localization of phosphorylation sites. On the other
hand, the lysine-phosphorylated peptides are relatively unstable
in a vacuum, and localization of the phosphorylation site, even
using the ECD method, should be done with caution.
FUNDING
This work was supported by the Ministry of Science and Higher
Education [grant number N401 2334 34].
REFERENCES
1 Raggiaschi, R., Gotta, S. and Terstappen, G. C. (2005)
Phosphoproteome analysis. Biosci. Rep. 25, 33–44
2 Cohen, P. (2002) The origins of protein phosphorylation. Nature
Cell Biol. 4, E127–E130
3 Marks, F. (1996) Protein Phosphorylation. Wiley-VCH, New York
4 Smith, J. R., Olivier, M. and Greene, A. S. (2007) Relative
quantification of peptide phosphorylation in a complex mixture
using 18O labeling. Physiol. Genomics 31, 357–363
5 McLachlin, D. T. and Chait, B. T. (2001) Analysis of phosphor ylated
proteins and peptides by mass spectrometry. Curr. Opin. Chem.
Biol. 5, 591–602
6 Besant, P. G. and Attwood, P. V. (2005) Mammalian histidine
kinases. Biochim. Biophys. Acta 1754, 281–290
7 Hoch, J. A. (2000) Two-component and phosphorelay signal
transduction. Curr. Opin. Microbiol. 3, 165–170
8 Thomason, P. and Kay, R. (2000) Eukaryotic signal transduction via
histidine-aspartate phosphorelay. J. Cell Sci. 113, 3141–3150
9 Attwood, P. V., Piggott, M. J., Zu, X. L. and Besant, P. G. (2007)
Focus on phosphohistidine. Amino Acids 32, 145–156
10 Zu, X. L., Besant, P. G., Imhof, A. and Attwood, P. V. (2007) Mass
spectrometric analysis of protein histidine phosphorylation. Amino
Acids 32, 347–357
11 Wind, M., Wegener, A., Kellner, R. and Lehmann, W. D. (2005)
Analysis of CheA histidine phosphorylation and its influence on
protein stability by high-resolution element and electrospray mass
spectrometry. Anal. Chem. 77, 1957–1962
12 Kleinnijenhuis, A. J., Kjeldsen, F., Kallipolitis, B., Haselmann, K. F.
and Jensen, O. N. (2007) Analysis of histidine phosphorylation
using tandem MS and ion-electron reactions. Anal. Chem. 79,
7450–7456
13 Besant, P. G. and Attwood, P. V. (2009) Detection and analysis of
protein histidine phosphorylation. Mol. Cell. Biochem. 329,
93–106
14 Besant, P. G., Attwood, P. V. and Piggott, M. J. (2009) Focus on
phosphoarginine and phospholysine. Curr. Protein Pept. Sci. 10,
536–550
15 Matthews, H. R. and Huebner, V. D. (1984) Nuclear protein
kinases. Mol. Cell. Biochem. 59, 81–99
16 Wakim, B. T. and Aswad, G. D. (1994) Ca2+-calmodulin-dependent
phosphorylation of arginine in histone 3 by a nuclear kinase from
mouse leukemia cells. J. Biol. Chem. 269, 2722–2727
17 Uda, K., Fujimoto, N., Akiyama, Y., Mizuta, K., Tanaka, K.,
Ellington, W. R. and Suzuki, T. (2006) Evolution of the arginine
kinase gene family. Comp. Biochem. Physiol. D 1, 209–218
18 Sikorska, M. and Whitfield, J. F. (1982) Isolation and purification of
a new 105 kDa protein kinase from rat liver nuclei. Biochim.
Biophys. Acta 703, 171–179
19 Matthews, H. R. (1995) Protein kinases and phosphatases that
act on histidine, lysine or arginine residues in eukaryotic proteins:
a possible regulator of the mitogen-activated protein kinase
cascade. Pharmacol. Ther. 67, 323–350
20 Hiraishi, H., Yokoi, F. and Kumon, A. (1998) 3-Phosphohistidine
and 6-phospholysine are substrates of a 56-kDa inorganic
pyrophosphatase from bovine liver. Arch. Biochem. Biophys. 349,
381–387
21 Yan, J. X., Packer, N. H., Gooley, A. A. and Williams, K. L. (1998)
Protein phosphorylation: technologies for the identification of
phosphoamino acids. J. Chromatogr. A 808, 23–41
22 Syrstad, E. A. and Turecek, F. (2005) Toward a general mechanism
of electron capture dissociation. J. Am. Soc. Mass. Spectrom. 16,
208–224
23 Stefanowicz, P., Kijewska, M. and Szewczuk, Z. (2009) Sequencing
of peptide-derived Amadori products by the electron induced
dissociation method. J. Mass. Spectrom. 44, 1047–1052
24 Shi, S. D., Hemling, M. E., Carr, S. A., Horn, D. M., Lindh, I. and
McLafferty, F. W. (2001) Phosphopeptide/phosphoprotein mapping
by electron capture dissociation mass spectrometry. Anal. Chem.
73, 19–22
25 Sweet, S. M., Bailey, C. M., Cunningham, D. L., Heath, J. K. and
Cooper, H. J. (2009) Large scale localization of protein
phosphorylation by use of electron capture dissociation mass
spectrometry. Mol. Cell. Proteomics 8, 904–912
26 Stefanowicz, P., Petry-Podgorska, I., Kowalewska, K., Jaremko, L.,
Jaremko, M. and Szewczuk, Z. (2010) Electrospray ionization mass
spectrometry as a method for studying the high-pressure
denaturation of proteins. Biosci. Rep. 30, 91–99
..........................................................................................................................................................................................................................................................................................................................................................................
442 C
The Authors Journal compilation C
2010 Biochemical Society
© 2010 The Author(s)
The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Detection of phospholysine in peptides
27 Rand, K. D. and Jørgensen, T. J. D. (2007) Development of a
peptide probe for the occurrence of hydrogen (H/H) scrambling
upon gas phase fragmentation. Anal. Chem. 79, 8686–8693
28 Wei, Y. F. and Matthews, H. R. (1991) Identification of
phosphohistidine in proteins and purification of protein-histidine
kinases. Methods Enzymol. 200, 388–414
29 Stokes, H. N. (1893) On diamidoor thophosphoric and
diamidotrihydroxylphosphoric acids. Am. Chem. J. 15,
198–214
30 Hall, K. U., Collins, S. P., Gamm, D. M., Massa, E., DePaoli-Roach,
A. A. and Uhle, M. D. (1999) Phosphorylation-dependent inhibition
of protein phosphatase-1 by G-substrate. J. Biol. Chem. 274,
3485–3495
31 Glass, D. B. (1983) Differential responses of cyclic
GMP-dependent and cyclic AMP-dependent protein kinases to
synthetic peptide inhibitors. Biochem. J. 213, 159–164
32 House, C. and Kemp, B. E. (1987) Protein kinase C contains a
pseudosubstrate prototope in its regulatory domain. Science 238,
1726–1728
33 Alexander, D. R., Graves, J. D., Lucas, S. C., Cantrell, D. A. and
Crumpton, M. J. (1990) A method for measuring protein kinase C
activity in permeabilized T lymphocytes by using peptide
substrates. Evidence for multiple pathways of kinase activation.
Biochem. J. 268, 303–308
34 Kelleher, N. L., Zubarev, R. A., Bush, K., Furie, B., Furie, B. C.,
McLafferty, F. W. and Walsh, C. T. (1999) Localization of labile
posttranslational modifications by electron capture dissociation:
thecaseofγ-carboxyglutamic acid. Anal. Chem. 71,
4250–4253
35 Pogliani, L., Ziessow, D. and Kr¨
uger, Ch. (1977) Conformational
study of phosphoserine in aqueous solutions. II - 1H n.m.r. results.
Org. Magn. Res. 10, 26–30
36 Raeck, C. and Berger, S. (2007) A 2D NMR method to study
peptide phosphorylation. Anal. Bioanal. Chem. 389, 2161–2165
37 Isab, A. A., Hussain, M. S., Akhtar, M. N., Wazeer, M.I.M. and
Al-Arfaj, A. R. (1999) 13C, 15 N and 31P NMR studies of the
disproportination of cyanogold(I) complexes: [R3PAu 13C15 N].
Polyhedron 18, 1401–1409
38 Lindon, J. C., Baker, D. J., Farrant, R. D. and Williams, J. M. (1986)
1H, 13C and 31 P n.m.r. spectra and molecular conformation of
myo-inositol 1,4,5-trisphosphate. Biochem. J. 233, 275–277
39 Varga-Defterdarovi´
c, L. and Hrlec, G. (2004) Synthesis and
intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related
6-O-glucopyranose esters. Carbohydr. Res. 339, 67–75
40 Ruman, T., Długopolska, K., Jurkiewicz, A., Rut, D., Fr ˛aczyk, T.,
Cie´
sla, J., Le´
s, A., Szewczuk, Z. and Rode, W. (2010)
Thiophosphorylation of free amino acids and enzyme protein by
thiophosphoramidate ions. Bioorg. Chem. 38, 74–80
Received 18 December 2009/26 January 2010; accepted 9 February 2010
Published as Immediate Publication 9 February 2010, doi 10.1042/BSR20090167
..........................................................................................................................................................................................................................................................................................................................................................................
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The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
... pHis is relatively stable under basic conditions (pH [10][11][12], and unstable under neutral and acidic conditions [17], and therefore Myo-pHis was buffer exchanged into 0.1 M Na 2 CO 3 /NaHCO 3 , at pH 10.8 (which also removed any unreacted potassium phosphoramidate), and subsequently concentrated and analyzed by 31 P NMR spectroscopy. Multiple pHis residue signals were observed in the chemical shift range of -4.40 to-5.76 ppm ( Fig 1A); additionally we observed an inorganic phosphate (Pi) signal at 2.57 ppm and a signal at 8.27 ppm which is discussed below and shown to derive from pLys [21] using an HMBC experiment (Fig 1B). ...
... The standard was contained in Norell1 high throughput 3 mm NMR sampling tubes. The capillary contained 2 or 4 mM triphenylphosphine oxide, 10 mol % chromium(III) acetylacetonate, dissolved in 150 μL of deuterated chloroform (CDCl 3 ). 1 H-31 P HMBC spectra were acquired on a Bruker AVANCE III 400 spectrometer, using 1664 scans for each of 128 increments over an acquisition window of 9.7 kHz and 3.6 kHz (2 k points) in F1 and F2 respectively and optimized for a long-range coupling constant of 10 Hz [19,21]. pSer, pThr, pTyr, 3-pHis, 1-pHis, and inorganic phosphate NMR spectra were recorded at room temperature. ...
... 10% (v/v) D 2 O was added to the sample before analysis by 31 P NMR spectroscopy. [21] and phosphorylate Lys under more basic conditions [21]. The above 31 P NMR spectrum suggests under the conditions used potassium phosphoramidate can phosphorylate other nucleophiles. ...
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Thesis
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Kovalente posttranslationale Modifikationen (PTMs) beeinflussen die Struktur und Funktion von Proteinen. Zu den bedeutendsten PTMs zählt die Proteinphosphorylierung. Labile Phosphorylierungen an Cystein- und Lysinresten, sowie Pyrophosphorylierungen an Serin- und Threoninbausteinen sind vermehrt in den Fokus der Wissenschaft gerückt. Trotz großer Fortschritte auf dem Gebiet der Massenspektrometrie (MS) bleibt die Analyse dieser empfindlichen Modifikationen mittels Tandem-MS eine große Herausforderung. In der vorliegenden Arbeit wird gezeigt, dass Elektronentransferdissoziation (ETD) in Kombination mit zusätzlicher HCD Aktivierung (EThcD) in der Lage ist, Peptide mit labilen Phosphorylierungen in der Seitenkette unter Erhalt der Modifikation zu fragmentieren. In verschiedenen proteomischen Ansätzen wird demonstriert, dass EThcD eine zweifelsfreie Identifizierung natürlich vorkommender Cysteinphosphorylierungen ermöglicht. Darüber hinaus wurde unter dem Gesichtspunkt der Labilität von Lysinphosphorylierungen ein bottom-up-Phosphoproteomikansatz etabliert. Das MS-Verfahren beruht auf der Generierung eines diagnostischen Phospholysinimmoniumions, welches im zweiten Schritt die Erfassung eines zusätzlichen EThcD-Spektrums desselben Precursorions veranlässt (triggert). Darüber hinaus wird im Zuge dieser Arbeit gezeigt, dass sich pyrophosphorylierte Peptide unter CID-Bedingungen in ihrem Neutralverlustmuster von isobaren diphosphorylierten Peptiden unterscheiden. Dieses Verhalten stellt einen Schlüsselschritt in einer neutralverlustgetriggerten EThcD Methode dar, welche die zweifelsfreie Identifizierung von Pyrophosphorylierungen ermöglicht. Darauf basierend konnten in Hefezellen und humanen embryonalen Nierenzellen die ersten Proteinpyrophosphorylierungen, einer neuen endogenen posttranslationalen Modifikation, nachgewiesen werden.
Article
Full-text available
Protein phosphorylation is a common signaling mechanism in both prokaryotic and eukaryotic organisms. Whilst serine, threonine and tyrosine phosphorylation dominate much of the literature there are several other amino acids that are phosphorylated in a variety of organisms. Two of these phosphoamino acids are phosphoarginine and phospholysine. This review will focus on the chemistry and biochemistry of both phosphoarginine and phospholysine. In particular we focus on the biological aspects of phosphoarginine as a means of storing and using metabolic energy (in place of phosphocreatine in invertebrates), the chemistry behind its synthesis and we examine the chemistry behind its highenergy phosphoramidate bond. In addition we will be reporting on the incidence of phosphoarginine in mammalian cells. Similarly we will be reviewing the current findings on the biology and the chemistry of phospholysine and its involvement in a variety of biological systems.
Book
Protein phosphorylation is a key mechanism in cellular signaling. This volume presents a state-of-the-art survey of one of the most rapidly developing fields of biochemical research. Written by leading experts, it presents the latest results for some of the most important cellular pathways. Color plates illustrate structural or functional relationships, numerous references provide links to the original literature. © VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1996. All rights reserved.
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Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.
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Article
Arginine kinase (AK), catalyzing the reversible transfer of phosphate from MgATP to arginine yielding phosphoarginine and MgADP, is widely distributed throughout the invertebrates and is also present in certain protozoa. Typically, these proteins are found as monomers targeted to the cytoplasm, but true dimeric and contiguous dimeric AKs as well as mitochondrial AK activities have been observed. In the present study, we have obtained the sequences of the genes for AKs from two distantly related molluscs-the cephalopod Nautilus pompilius and the bivalve Crassostrea gigas. These new data were combined with available gene structure data (exon/intron organization) extracted from EST and genome sequencing project databases. These data, comprised of 23 sequences and gene structures from Protozoa, Cnidaria, Platyhelminthes, Mollusca, Arthropoda and Nematoda, provide great insight into the evolution and divergence of the AK family. Sequence and phylogenetic analyses clearly show that the AKs are homologous having arisen from some common ancestor. However, AK gene organization is highly divergent and variable. Molluscan AK genes typically have a highly conserved six-exon/five-intron organization, a structure that is very similar to that of the platyhelminth Schistosoma mansoni Arthropod and nematode AK genes have fewer introns, while the cnidarian and protozoan genes each display unique exon/intron organization when compared to the other AK genes. The non-conservative nature of the AK genes is in sharp contrast to the relatively high degree of conservation of intron positions seen in a homologous enzyme creatine kinase (CK). The present results also show that gene duplication and subsequent fusion events forming unusual two-domain AKs occurred independently at least four times as these contiguous dimers are present in Protozoa, Cnidaria, Platyhelminthes and Mollusca. Detailed analyses of the amino acid sequences indicate that two AKs (one each from Drosophila and Caenorhabditis) have what appear to be N-terminal mitochondrial targeting sequences, providing the first evidence for true mitochondrial AK genes. The AK gene family is ancient and the lineage has undergone considerable divergence as well as multiple duplication and fusion events.
Article
In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of (31)P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of (31)P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His(26)) or lysine (Lys(33)) side-chain.
Article
Protein histidine phosphorylation is well established as an important part of signalling systems in bacteria, fungi and plants and there is growing evidence of its role in mammalian cell biology. Compared to phosphoserine, phosphothreonine and phosphotyrosine, phosphohistidine is relatively labile, especially under the acidic conditions that were developed to analyse protein phosphorylation. In recent years, there has been an increasing impetus to develop specific methods for the analysis of histidine phosphorylation and assay of histidine kinase activity. Most recently attention has focussed on the application of mass spectrometry to this end. This review provides an overview of methods available for the detection and analysis of phosphohistidine in phosphoproteins, with particular emphasis on the application of mass spectrometric techniques.