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Anal Bioanal Chem

DOI 10.1007/s00216-010-4128-3


Arsenic-induced protein phosphorylation changes

in HeLa cells
Orkun Alp & Edward J. Merino & Joseph A. Caruso

Received: 9 June 2010 / Revised: 11 August 2010 / Accepted: 11 August 2010

# Springer-Verlag 2010

Abstract Arsenic is well documented as a chemotherapeu- P signal vs. time via ICP-MS. SEC-ICP-MS fractions are
tic agent capable of inducing cell death while at the same collected and then separated by the nano-LC-CHIP/ITMS
time is considered a human carcinogen and an environ- system for peptide determination. Spectrum Mill and
mental contaminant. Although arsenic toxicity is well MASCOT protein database search engines are used for
known and has formed an impressive literature over the protein identification. Several phosphorylation sites and
time, little is known about how its effects are exerted at the proteins related to post-translational modifications are also
proteome level. Protein phosphorylation is an important identified.
post-translational modification involved in the regulation of
cell signaling and likely is altered by arsenic treatment. Keywords Bioanalytical methods . Cell systems/single-cell
Despite the importance of phosphorylation for many analysis . Mass spectrometry/ICP-MS . Speciation . HPLC .
regulatory processes in cells, the identification and charac- Genomics/proteomics
terization of phosphorylation, as effected by arsenic
through mass spectrometric detection, are not fully studied.
Here, we identify phosphorylated proteins, which are Introduction
related to post-translational modifications after phenylarsine
oxide (PAO) inoculation to HeLa cells. PAO was chosen Certain arsenic species are cytotoxic substances, and it is
because of its high cytotoxicity, measured earlier in these known that among the arsenic species, cytotoxicity of As
labs. In this study, size exclusion chromatography coupled (III) is higher than As(V) [1]. Phenylarsenic compounds
to inductively coupled plasma mass spectrometry (SEC- may play a significant role in environmental contaminations
ICP-MS) is used to establish several molecular weight caused by residues of chemical warfare agents (CWAs) [2,
fractions with phosphorylated proteins by monitoring 31 3]. The arsenic-based warfare compounds diphenylarsine
chloride (CLARK I), diphenylarsine cyanide (CLARK II),
phenarsazine chloride (Adamsite), and phenylarsine
O. Alp dichloride (Pfiffikus) were produced during World War
Analytical Chemistry Department, Faculty of Pharmacy,
I–II in large scale [4]. Arsenic-based CWAs degrade to
Gazi University,
06330 Ankara, Turkey diphenylarsinic acid, phenylarsonic acid, and phenylarsine
oxide (PAO). An earlier study has shown that PAO is the
O. Alp : J. A. Caruso most cytotoxic of the three for monkey kidney cells under
University of Cincinnati/Agilent Technologies Metallomics
similar experimental conditions [5]. At locations where
Center of the Americas, Department of Chemistry,
University of Cincinnati, CWAs were deposited, degradation products often contam-
Cincinnati, OH 45221-0172, USA inate the environment, resulting in arsenic levels of, for
example, 923 mgkg−1 soil on average [6]. The cytotoxic
E. J. Merino : J. A. Caruso (*)
effects of arsenic species are well documented in the
Department of Chemistry, University of Cincinnati,
Cincinnati, OH 45221-0172, USA literature, and the cytotoxicity of arsenic degradation
e-mail: products is due to differences in cellular response [5, 7–9].
O. Alp et al.

However, beyond the cytotoxicity studies, there are numer- ablation inductively coupled plasma technique to interro-
ous molecular-level changes that may be monitored in the gate electrophoresis gels and gel blots. This initial study
cell and these include post-translational modifications using gel blots was refined by Wind et al. [23] who added a
(PTMs). Although many studies have been published dealing washing step to eliminate non-covalently bound phospho-
with structure elucidation and quantification of low molec- rus as interference in the phosphoprotein measurement.
ular weight arsenic compounds in biological samples, Recently, elemental mass spectrometry was introduced
binding of arsenic to larger biomolecules, such as polypep- as an alternative tool for spotting of phosphorylation sites
tides and proteins, is less often studied [10, 11]. The and determination of phosphorylation stoichiometry[24].
biochemical mechanisms responsible for these effects caused This method is based on coupling capillary liquid chroma-
by arsenic remain unclear [12] but may be mediated by the tography (μLC) with ICP-MS and was successfully applied
binding of trivalent arsenicals (such as PAO) to thiol groups both on the peptide and protein level [25, 26]. The element-
in proteins, thereby changing the conformation of these selective ICP-MS detector allows for screening of
proteins and altering their functions. If some of the affected phosphorus-containing compounds in the LC eluate, and
proteins are responsible for cellular repair of DNA damage, the signal intensity is directly proportional to the phospho-
for example, the inhibition of these proteins could lead to rus content. By taking appropriate fractions as is or by
carcinogenesis. Recent studies have shown binding of combining fractions at the same retention time, preconcen-
trivalent arsenicals to cysteines in proteins [13, 14]. tration is achieved, which is highly useful for the molecular
Protein phosphorylation, one of the most important post- MS identification experiment in the event of low copy
translational protein modifications, plays an important role numbers of proteins.
in regulating cellular processes through cell signaling. Human cervical cancer HeLa cells are a known model
Thus, characterization of protein phosphorylation is of system and have been shown to uptake arsenic species well
great interest for understanding cell regulation mechanisms. [27, 28]. Therefore, HeLa cells were used as target cells
Protein phosphorylation analysis can be performed at two with our focus to study the cytotoxic effect of PAO on
levels. One is the proteome level, and the other is the HeLa cells by contemporaneously determining protein
individual protein level. A proteome sample, typically total phosphorylation and phosphorylation changes in toxified
cell lysate, is highly complex. Therefore, the primary issue cells. PAO was chosen since it has been shown to be highly
for phosphoproteome analysis is to reduce the sample cytotoxic from an earlier study [5], and with the conditions
complexity. Thus, specific enrichment of phosphopeptides in this study it had a greater cytotoxic effect than As3+.
from the protein mixture digest and efficient separation of
the enriched phosphopeptides prior to mass spectrometric
analysis is crucial for an effective phosphoproteome Experimental
analysis [15, 16]. In comparison with large-scale phosphor-
ylation analysis at the proteome level, phosphorylation site Reagents
mapping of individual phosphoproteins is of equal impor-
tance [17, 18]. The sample for the analysis of phosphory- All reagents were of analytical reagent grade. Unless stated
lation sites on an individual protein is not as complex since otherwise, all the solutions were prepared in 18 MΩcm−1
only one protein is presented in the sample. For many doubly deionized water (Sybron Barnstead, Boston, MA,
cases, in biological applications, only a trace amount of USA). Tris(hydroxymethyl)aminomethane (Acros Organ-
protein is available for analysis. Therefore, an important ics, Morris Plains, NJ, USA) was used as buffer for liquid
issue for phosphorylation analysis of individual proteins is chromatography. The buffer pH was adjusted to 7.5 using
to improve the detection capabilities. hydrochloric acid (Pharmco Products, Inc., Brookfield, CT,
There are established methods for the detection of USA). PAO was obtained from Alfa Aesar, Lancaster, UK.
phosphoproteins, the majority based on separation by PAO stock solution was prepared in 50% dimethyl
electrophoresis or chromatography followed by radiochem- sulfoxide (DMSO; Fisher Scientific, Fairlawn, NJ, USA)
ical detection or molecular mass spectrometry [19, 20]. As to obtain a stock solution of 200 mmoll−1. The high-
an addition to molecular mass spectrometry, elemental mass performance liquid chromatography (HPLC) solvents,
spectrometry based on inductively coupled plasma mass water and acetonitrile (ACN), were of high purity and
spectrometry (ICP-MS) has been proposed for the determi- purchased from Burdick and Jackson (Muskegon, MI).
nation of phosphoproteins. In one of the earliest studies, Sequence-grade-modified trypsin and the acetic acid buffer
Wind et al. [21] described the combination of capillary were obtained from Promega (Madison, WI). Formic acid
liquid chromatography and ICP-MS to identify phosphor- (FA) was purchased from Agilent (USA). Dithiothreitol,
ylated proteins. As a complimentary approach to phospho- iodoacetamide, protein standards β-casein, bovine serum
protein measurements, Marshall et al. [22] utilized the laser albumin (BSA), thyroglobulin, conalbumin, myoglobin,
Arsenic-induced protein phosphorylation changes in HeLa cells

and B12 were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Urea and ammonium bicarbonate were from

Cell viability, % of Control

Fisher Scientific (Fairlawn, NJ, USA).
Cells and culture conditions

The HeLa cell lines were available at our labs at University

of Cincinnati. HeLa cells were grown in 25-cm2 flasks and
maintained in Dulbecco’s Modified Eagle’s Medium 20
(DMEM: Fisher Scientific, Fairlawn, NJ, USA) supple-
mented with 10% fetal bovine serum (Fisher Scientific, 0
Fairlawn, NJ, USA) at 37 °C with 5% CO2 in a humidified 0.01 0.1 1 5 10

atmosphere. Subculture passages were performed every PAO concentration, µmol l-1

3 days using trypsin–ethylenediaminetetraacetic acid 3 hours 6 hours 24 hours

(EDTA): 0.05% trypsin 0.53 mM EDTA×4Na (Gibco Fig. 1 Dose-dependent viability changes in PAO-exposed HeLa cells.
Invitrogen Corporation, Carlsbad, CA, USA). All PAO Data are shown as percentages of the control value. Each value
solutions were made up fresh by diluting the stock solution represents the average ± SD of quadruplicate wells
in DMEM media prior to dosing. PAO was added to cell
culture flasks at the indicated times and concentrations nebulizer, a Peltier-cooled spray chamber (2 °C), and a
below prior to harvest. After treating the cells with PAO, shield torch constitute the sample introduction system under
mitochondrial function was measured using the tetrazolium standard plasma conditions. Instrument conditions are
salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium given in Table 1.
bromide (MTT) as an indicator of cell viability.
High-performance liquid chromatography
Cytotoxicity assay (MTT assay)
Chromatographic separations were carried out with an
HeLa cells were seeded in 24-well plates at a density of Agilent 1100 liquid chromatograph (Agilent Technologies,
1×104 ml−1 and incubated for 24 h before adding various
concentrations of PAO (0.01, 0.1, 1, 5, and 10 μmoll−1). Table 1 Operating conditions for ICP-MS, HPLC, and nano-LC-CHIP/
Cells were exposed to PAO at indicated concentrations for
3, 6, and 24 h. ICP-MS parameters
A working solution of MTT reagent was dissolved in Forward power, W 1,500
1-ml phosphate saline buffer (PBS; Gibco Invitrogen Plasma gas flow rate, lmin−1 15.0
Corporation, Carlsbad, CA, USA) at a concentration of Carrier gas flow rate, lmin−1 1.05
5 mgml−1 and diluted to a volume of 50 ml with media. Isotope monitored 31
After removing the media in each well at the end of Collision gas (He) flow rate, mlmin−1 3.6
exposure time, 1 ml of MTT–media solution was placed in Quadrupole bias, V −16
each well and incubated for 3 h. After removing the media– Octopole bias, V −18
MTT solution, 0.5 ml DMSO was added to each well. Then Energy discrimination voltage +2
the culture plate was placed on a micro-plate reader and the SEC-HPLC parameters
absorbance measured at 550 nm with background correc- Column TSK-GEL G3000SW
tion. Results are shown in Fig. 1. Mobile phase 30 mM Tris–HCl buffer,
pH 7.5
Instrumentation Flow rate, mlmin−1 0.5
Injection volume, μl 50
Inductively coupled plasma mass spectrometry Nano-LC-CHIP/ITMS parameters
Drying gas, N2, 4 lmin-1
An Agilent 7500ce ICP-MS (Agilent Technologies, Santa Temperature 300°C
Clara, CA, USA) was used for phosphorus (31P) detection. MS capillary voltage 1,900 V
It is equipped with an octopole cell and can be operated Skimmer 30 V
with or without the collision/reaction gas. Helium (He) was Capillary exit 128 V
used as collision gas at a flow rate of 3.6 mlmin−1 Trap drive 75 V
throughout the experiments. A conventional Meinhard
O. Alp et al.

Santa Clara, CA, USA) equipped with a binary HPLC criteria were used for validation: minimum scores for
pump, an autosampler, a vacuum degasser system, a spectra resulting from fragmentation of 1+, 2+, 3+, and 4+
temperature column compartment, and a diode array for ions were 8, 7, 9, and 9 respectively. Scored peak
detector. The outlet of the UV detector was connected to intensity (SPI) value was set to at least 70%. Also, in order
sample inlet of the ICP-MS nebulizer using 0.25-mm i.d. to minimize false-positive results, only spectra with a
polyether ether ketone tubing of 30 cm in length. For size reversed score that was at least twice smaller than the real
exclusion chromatography (SEC), a TSK-GEL G3000SW score were taken into account. The data obtained from the
(7.5 mm×300 mm, 10 μm) (Tosoh Bioscience LLC, Tokyo, nano-LC-CHIP system were also searched using MASCOT
Japan) was used. The SEC column was calibrated with a (Matrix Science, London, UK, online public version).
protein mixture of thyroglobulin (670 kDa), conalbumin Swiss-Prot protein database was selected for both search
(77 kDa), myoglobin (17 kDa), and B12 (1.3 kDa), and they engines to compare the method confidence in peptide
eluted at 7, 10, 13, and 17 min, respectively. UV absorption assignments.
was monitored at 280 nm. The instrumental conditions are
shown in Table 1. Sample preparation and phosphoprotein fraction
determination by SEC-ICP-MS
PAO was utilized since it produced the maximal toxifica-
All electrospray experiments were done on an Agilent 6300 tion in the shortest time period, consistent with the project
Series HPLC-CHIP/Ion Trap XCT system (Agilent Tech- goals of assessing changes in phosphorylation as a
nologies, Santa Clara, CA). The Agilent 1200 LC equipped function of cellular toxicity. PAO treatment was initiated
with both a capillary and nano-pump was used for loading when the cells were in 80% confluency as estimated by the
and flushing the Agilent chip nanocolumn. The chip used microscopic images. Cells were treated with PAO
contained a Zorbax 300SB C18 enrichment column (0.01–10 μmoll−1) for 3 h and at the end of the incubation
(4 mm×75 μm, 5 μm) and a Zorbax 300SB C18 analytical period cells were washed twice with PBS in order to
column (150 mm×75 μm, 5 μm). Sample loading onto the remove the media residue. After removal of the superna-
enrichment column was set at a flow rate of 3 μlmin−1 with tant, the cells were harvested by trypsin–EDTA and then
a 97:3 ratio of solvent A and B (A 100% H2O, 0.1% FA; B lysed by M-Per protein extraction buffer (Fisher Scientific,
90% ACN, 10% H2O, 0.1% FA). After loading the Fairlawn, NJ, USA). The sample was centrifuged at
enrichment column, the flush is switched by on-chip 13,000×g for 10 min, and the supernatant was taken and
microfluidics to the analytical column at a flow rate of analyzed by SEC-ICP-MS.
0.3 μL/min with the following gradient conditions:
0–3 min, 3% B; 3–80 min, 60% B; 80–82 min, 60% B; Sample preparation for nano-LC-CHIP/ITMS
82–83 min, 95% B; 83–85 min, 95% B; 85–87 3% B,
followed by 10 min of column re-equilibration. The MS β-casein and BSA were used as model proteins to ensure
parameters used for phosphoprotein identification were confidence of the method. Tryptic digestion of β-casein and
shown in Table 1. The target number of ions was 500,000 BSA was performed as follows: 1 mg of β-casein and BSA
with a maximum accumulation time of 300 ms. The MS was dissolved in 1 ml 50 mM NH4HCO3. A 15-μl portion
scan range was 50–2,200m/z in standard enhanced scan of protein mixture was pipetted from the stock solution, and
mode. Five parent ions were selected and isolated by the urea was added to the dissolved protein mixture at a
instrument, producing MS2 and MS3 spectra by collision- concentration of 6 M. Then 5 μl of 45 mM dithiothreitol
induced dissociation (CID) with He gas and fragmentation was added to reduce the protein, and the solution was
amplitude was set to 1.30 V. incubated at 50 °C for 30 min. After the solution was
cooled to room temperature, 5 μl of 100 mM iodoacetamide
Peptide identification was added, after which, it was left in the dark at room
temperature for 30 min. Sample was diluted with DDI
Database searching was performed on Spectrum Mill water in order to have a final concentration of urea less than
(Rev. A. 03.02) (Agilent Technologies, Santa Clara, CA). 1 M. For digestion, 20 μg of trypsin was dissolved in 200
The selected parameters used for the searches were data μl of 50 mM acetic acid (as a resuspension buffer). From
searched against the Swiss-Prot database; taxonomy: homo this solution, 5 μl of the trypsin solution was added to the
sapiens; enzyme: trypsin, two missed cleavages; variable protein mixture and then incubated at 37 °C overnight. To
modification: phosphorylation (threonine, serine, tyrosine inhibit the trypsin activity, 3% formic acid was added to the
or T, S, Y, respectively). The following autovalidation samples (final FA concentration was 0.3%). The samples
Arsenic-induced protein phosphorylation changes in HeLa cells

Table 2 Spectrum Mill and MASCOT results of β-casein and BSA, method test samples

Spectrum Mill MASCOT

# of matched Summed Mean MW, Da Protein name # of matched Summed MW, Da Protein name
peptide scorea SPIb peptide scorea

1 1 22 6.52E+06 25,107.5 β-casein (bovine) 1 76 25,148 β-casein (bovine)

2 12 116 1.93E+08 69,293.9 Serum albumin (bovine) 19 312 71,244 Serum albumin (bovine)
Sum of scores of each identified peptide
Scored peak intensity

were passed through 0.22-μm filters (Agilent Technologies, Results and discussion
Santa Clara, CA) prior to analysis. Two microliters of
digested protein standard was injected to the nano-LC- Cytotoxicity of PAO
CHIP/ITMS system using the optimized conditions, and the
data obtained were then searched using both Spectrum Mill HeLa cells were exposed to five different concentrations of
and MASCOT for identification. Tryptic digestion of cell PAO: 0.01, 0.1, 1, 5, and 10 μmoll−1; and three different
lysate samples was achieved in the same way. exposure times were utilized. Since reduction of MTT can
The results for β-casein obtained from both search only occur in metabolically active cells, the level of activity
engines gave the same singly phosphorylated peptide on is a measure of cell viability.
serine (K)FQsEEQQQTEDELQDK(I), and the phosphory- PAO was cytotoxic to HeLa cells. When PAO was
lation was on site 50S of the protein. The molecular ion at incubated at a concentration of 0.01 μmoll−1, viability of
2,060 Da and MW of 25,107.5 Da were reported for cells did not differ significantly throughout the studied
β-casein. BSA search results had greater confirmation over growth period. On the other hand, by increasing the
the β-casein results. MASCOT gave 19 matched peptides concentration of PAO to 0.1 μmoll−1, cell death signifi-
where Spectrum Mill gave 12 matched peptides, and protein cantly increased (75%±3) with 24-h exposure. It has been
MW of 71,244 Da was reported as was serum albumin for the suggested that the toxic effects of arsenic species with
protein name. The results are summarized in Table 2. phenyl groups are achieved more quickly than other arsenic

Fraction 4

1 µmol l PAO
400000 35000
350000 30000
Counts, P

(A) 20000
Counts, P

Fraction 3

Fraction 2

Fraction 5

Fraction 1

200000 10000

100000 7.0 9.3 11.6 13.9 16.2
Time, min

0.0 6.1 12.2 18.4 24.5
Time, min

Fig. 2 a Comparison of the SEC-ICP-MS chromatograms of control and 1 μmoll−1 PAO exposure time 3 h (measured at m/z 31
P), b enlarged
view of the SEC-ICP-MS chromatogram
O. Alp et al.

GC-rich sequence DNA-binding factor homolog (SEC fraction 2)

species possibly because the cell membrane may be more
permeable to As species with phenyl groups [5].

RAC-alpha serine/threonine protein kinase (SEC fraction 3)

Since the amount of phosphorylated proteins was

Succinyl-CoA ligase (GDP-forming) (SEC fraction 4)

expected to be very limited, 3 h of PAO exposure time

Src substrate cortactin (Amplaxin) (SEC fraction 3)

was chosen in order to assure ≥70% viable cells for SEC-
Ras GTPase-activating protein (SEC fraction 2)

Rho GTPase-activating protein (SEC fraction2)

ICP-MS experiments.

DNA polymerase kappa (SEC fraction 1)

Netrin receptor UNC5D (SEC fraction 2)

Endonuclease domain (SEC fraction 4)

SEC-ICP-MS results

Sphingosine kinase 2 (SEC fraction 3)

HeLa cells were incubated for 3 h with various concen-
trations of PAO (0.01–10 μmoll-1). When SEC-ICP-MS
Matrin-3 (SEC fraction 2)

chromatogram of 1 μmoll−1 PAO was compared with the

control group, significant differences were observed in SEC
chromatograms as shown in Fig. 2. The differences
between the chromatograms of control group and the
Matched parent Protein name

PAO-inoculated group are in the range of ∼1–100 kDa.

On the other hand, with up to 0.1 μmol l −1 PAO
concentration, the differences in the chromatograms are
insignificant when compared with control. According to
these results, PAO may exert its cytotoxic effects by either
mass (Da)




inhibiting protein phosphatases or stimulating protein


kinases, since these enzymes, respectively, are related with

Table 3 Results of phosphorylated proteins obtained from Spectrum Mill and MASCOT database search engines

dephosphorylation and phosphorylation events in cells.


Fraction collection at m/z=31, P

The SEC column effluent was collected as fractions for three



injections at time intervals that represented the elution of the


unidentified phosphorus species some of which are unidenti-

fied phosphorylated proteins. The time intervals for collection

were offset by 0.1 min, to compensate for the lag time from the


end of the HPLC column to the ICP-MS detection system.

After collection, the samples were lyophilized and reconsti-
tuted with 200 μl of 50 mM NH4HCO3 for determination of
protein amounts of each fraction. Protein concentrations
were determined by using Bio-Rad DC Protein Assay Kit to
estimate total protein concentration. The assay was per-

formed according to manufacturer’s instructions.

Phosphoprotein identification by nano-LC-CHIP/ITMS

Spectrum Mill MASCOT Variable

Protein identification of each fraction was performed by

S8s S9s






Spectrum Mill, and the results were compared with the

MASCOT protein database search engine to increase the
peptide assignment confidence. The results are summarized
in Table 3 for the first four fractions collected as shown in





Fig. 2. The scores reported in the description below belong

only to Spectrum Mill, but the score comparisons are

shown in Table 3. Note that the Spectrum Mill and
MASCOT scores cannot be directly compared, but the

scores reported for both search engines are valid scores.




The proteins reported are those which coincide with


molecular weight ranges associated with the SEC fractions.


Arsenic-induced protein phosphorylation changes in HeLa cells

Table 4 Spectrum Mill and MASCOT results of non-phosphorylated PTM-related proteins

Spectrum Mill MASCOT Sequence Matched parent Protein name

score score mass (Da)

1 19.28 74 (K)GADFLVTEVENGGSLGSK(K) 1,779.876 Pyruvate kinase isozymes M1/M2

2 19.09 82 (R)LPVVIGGLLDVDCSEDVIK(N) 2,041.089 Clathrin heavy chain 1
3 16.16 50 (K)TFTDCFNCLPIAAIVDEK(I) 2,113.993 Serine/threonine-protein phosphatase PP1
4 15.75 47 (K)VWLDPNETNEIANANSR(Q) 1,942.925 60S ribosomal protein L19
5 14.54 53 (R)YGINTTDIFQTVDLWEGK(N) 2,100.028 Transgelin-2
6 14.43 68 (R)YMIGVTYGGDDIPLSPYR(I) 2,016.974 Filamin-B
7 13.56 78 (K)STNGDTFLGGEDFDQALLR(H) 2,055.962 Stress-70 protein
8 12.22 64 (R)FDQLFDDESDPFEVLK(A) 1,943.891 Plasminogen activator inhibitor 1
RNA-binding protein

DNA polymerase kappa is the only result for fraction 1 ylation, and according to the protein search engines results,
with a score of 9.55. This protein is specifically involved in Amplaxin has a site modification at T410t. Amplaxin is a
DNA repair and plays an important role in translesion monomeric protein located in the cytoplasm of cells that
synthesis, where the normal high-fidelity DNA polymerases can be activated by external stimuli to promote polymeri-
cannot proceed and DNA synthesis stalls [29]. One of the zation and rearrangement of the actin cytoskeleton [36].
results for fraction 2 is the Ras GTPase-activating protein Actin is a globular protein found in all eukaryotic cells
(GAP), which is a target for protein tyrosine kinases of both which participates in many important cellular processes
the receptor and cytoplasmic classes and may serve to such as cell signaling and cell division. The other identified
integrate tyrosine kinase and Ras signaling pathways [30]. protein is sphingosine kinase 2 (SPHK2) which catalyzes
GAP phosphorylated exclusively on serine residues and the phosphorylation of sphingosine to form sphingosine 1
recruited to stress granules (SGs) upon either arsenite or phosphate. SPHK2 is another member of a growing class of
high-temperature treatment [31]. Another important protein sphingolipid kinases that may have novel functions such as
for post-translational modifications is Rho GTPase- apoptosis or inhibition of cellular proliferation [37], which
activating protein, identified with a high score of 9.63. suggests mediating the cancerous HeLa cell growth. RAC-
The Rho GTPase-activating proteins are one of the major alpha serine/threonine protein kinase involves several
classes of regulators of Rho GTPases found in all different cellular functions, including the control of cell
eukaryotes that are crucial in cell cytoskeletal organization, size and the regulation of survival and metabolism [38].
growth, differentiation, neuronal development, and synaptic Two phosphorylated proteins are observed for fraction 4 of
functions [32]. Netrin is another PTM-related protein, and the SEC-ICP-MS chromatogram, and one of them is
this protein is phosphorylated on cytoplasmic tyrosine endonuclease that has a modification site on Y78y. Its
residues and proteolytically cleaved by caspases during molecular function is catalysis of the hydrolysis of ester
apoptosis [33, 34]. Spectrum Mill results showed two linkages within nucleic acids and also it can interact non-
modifications on tyrosine (Y947y) and serine (S948s). covalently with metals and nucleic acids. The other
GC-rich sequence DNA-binding factor protein is involved phosphorylated protein is succinate-CoA ligase. It is a
in the regulation of transcription in biological process [34]. mitochondrial matrix enzyme and catalyzes the reversible
Matrin 3 is a nuclear matrix protein that has been conversion of succinyl-CoA and ADP or GDP to succinate
implicated in interacting with other nuclear proteins to and ATP or GTP [39]. Both database search engines did not
anchor hyperedited RNAs to the nuclear matrix. Matrin 3 give any valid results for fraction 5 as was expected, since
was identified as both a Ca2+-dependent CaM-binding its retention time is around 1 kDa. The results for identified
protein and a downstream substrate of caspases [35]. Src phosphoproteins are summarized in Table 3. Nevertheless,
substrate cortactin (Amplaxin), which is one of the results the major aim of this study is to identify peptide
for fraction 3, is related to the reversible protein phosphor- phosphorylation sites and possible proteins, and several
O. Alp et al.

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