Anal Bioanal Chem
DOI 10.1007/s00216-010-4128-3
ORIGINAL PAPER
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-
tic agent capable of inducing cell death while at the same
time is considered a human carcinogen and an environ-
mental contaminant. Although arsenic toxicity is well
known and has formed an impressive literature over the
time, little is known about how its effects are exerted at the
proteome level. Protein phosphorylation is an important
post-translational modification involved in the regulation of
cell signaling and likely is altered by arsenic treatment.
Despite the importance of phosphorylation for many
regulatory processes in cells, the identification and charac-
terization of phosphorylation, as effected by arsenic
through mass spectrometric detection, are not fully studied.
Here, we identify phosphorylated proteins, which are
related to post-translational modifications after phenylarsine
oxide (PAO) inoculation to HeLa cells. PAO was chosen
because of its high cytotoxicity, measured earlier in these
labs. In this study, size exclusion chromatography coupled
to inductively coupled plasma mass spectrometry (SEC-
ICP-MS) is used to establish several molecular weight
fractions with phosphorylated proteins by monitoring 31
O. Alp
Analytical Chemistry Department, Faculty of Pharmacy,
Gazi University,
06330 Ankara, Turkey
O. Alp : J. A. Caruso
University of Cincinnati/Agilent Technologies Metallomics
Center of the Americas, Department of Chemistry,
University of Cincinnati,
Cincinnati, OH 45221-0172, USA
E. J. Merino : J. A. Caruso (*)
Department of Chemistry, University of Cincinnati,
Cincinnati, OH 45221-0172, USA
e-mail: joseph.caruso@uc.edu
P signal vs. time via ICP-MS. SEC-ICP-MS fractions are
collected and then separated by the nano-LC-CHIP/ITMS
system for peptide determination. Spectrum Mill and
MASCOT protein database search engines are used for
protein identification. Several phosphorylation sites and
proteins related to post-translational modifications are also
identified.
Keywords Bioanalytical methods . Cell systems/single-cell
analysis . Mass spectrometry/ICP-MS . Speciation . HPLC .
Genomics/proteomics
Introduction
Certain arsenic species are cytotoxic substances, and it is
known that among the arsenic species, cytotoxicity of As
(III) is higher than As(V) [1]. Phenylarsenic compounds
may play a significant role in environmental contaminations
caused by residues of chemical warfare agents (CWAs) [2,
3]. The arsenic-based warfare compounds diphenylarsine
chloride (CLARK I), diphenylarsine cyanide (CLARK II),
phenarsazine chloride (Adamsite), and phenylarsine
dichloride (Pfiffikus) were produced during World War
I–II in large scale [4]. Arsenic-based CWAs degrade to
diphenylarsinic acid, phenylarsonic acid, and phenylarsine
oxide (PAO). An earlier study has shown that PAO is the
most cytotoxic of the three for monkey kidney cells under
similar experimental conditions [5]. At locations where
CWAs were deposited, degradation products often contam-
inate the environment, resulting in arsenic levels of, for
example, 923 mgkg−1 soil on average [6]. The cytotoxic
effects of arsenic species are well documented in the
literature, and the cytotoxicity of arsenic degradation
products is due to differences in cellular response [5, 7–9].

However, beyond the cytotoxicity studies, there are numer-
ous molecular-level changes that may be monitored in the
cell and these include post-translational modifications
(PTMs). Although many studies have been published dealing
with structure elucidation and quantification of low molec-
ular weight arsenic compounds in biological samples,
binding of arsenic to larger biomolecules, such as polypep-
tides and proteins, is less often studied [10, 11]. The
biochemical mechanisms responsible for these effects caused
by arsenic remain unclear [12] but may be mediated by the
binding of trivalent arsenicals (such as PAO) to thiol groups
in proteins, thereby changing the conformation of these
proteins and altering their functions. If some of the affected
proteins are responsible for cellular repair of DNA damage,
for example, the inhibition of these proteins could lead to
carcinogenesis. Recent studies have shown binding of
trivalent arsenicals to cysteines in proteins [13, 14].
Protein phosphorylation, one of the most important post-
translational protein modifications, plays an important role
in regulating cellular processes through cell signaling.
Thus, characterization of protein phosphorylation is of
great interest for understanding cell regulation mechanisms.
Protein phosphorylation analysis can be performed at two
levels. One is the proteome level, and the other is the
individual protein level. A proteome sample, typically total
cell lysate, is highly complex. Therefore, the primary issue
for phosphoproteome analysis is to reduce the sample
complexity. Thus, specific enrichment of phosphopeptides
from the protein mixture digest and efficient separation of
the enriched phosphopeptides prior to mass spectrometric
analysis is crucial for an effective phosphoproteome
analysis [15, 16]. In comparison with large-scale phosphor-
ylation analysis at the proteome level, phosphorylation site
mapping of individual phosphoproteins is of equal impor-
tance [17, 18]. The sample for the analysis of phosphory-
lation sites on an individual protein is not as complex since
only one protein is presented in the sample. For many
cases, in biological applications, only a trace amount of
protein is available for analysis. Therefore, an important
issue for phosphorylation analysis of individual proteins is
to improve the detection capabilities.
There are established methods for the detection of
phosphoproteins, the majority based on separation by
electrophoresis or chromatography followed by radiochem-
ical detection or molecular mass spectrometry [19, 20]. As
an addition to molecular mass spectrometry, elemental mass
spectrometry based on inductively coupled plasma mass
spectrometry (ICP-MS) has been proposed for the determi-
nation of phosphoproteins. In one of the earliest studies,
Wind et al. [21] described the combination of capillary
liquid chromatography and ICP-MS to identify phosphor-
ylated proteins. As a complimentary approach to phospho-
protein measurements, Marshall et al. [22] utilized the laser
O. Alp et al.
ablation inductively coupled plasma technique to interro-
gate electrophoresis gels and gel blots. This initial study
using gel blots was refined by Wind et al. [23] who added a
washing step to eliminate non-covalently bound phospho-
rus as interference in the phosphoprotein measurement.
Recently, elemental mass spectrometry was introduced
as an alternative tool for spotting of phosphorylation sites
and determination of phosphorylation stoichiometry[24].
This method is based on coupling capillary liquid chroma-
tography (μLC) with ICP-MS and was successfully applied
both on the peptide and protein level [25, 26]. The element-
selective ICP-MS detector allows for screening of
phosphorus-containing compounds in the LC eluate, and
the signal intensity is directly proportional to the phospho-
rus content. By taking appropriate fractions as is or by
combining fractions at the same retention time, preconcen-
tration is achieved, which is highly useful for the molecular
MS identification experiment in the event of low copy
numbers of proteins.
Human cervical cancer HeLa cells are a known model
system and have been shown to uptake arsenic species well
[27, 28]. Therefore, HeLa cells were used as target cells
with our focus to study the cytotoxic effect of PAO on
HeLa cells by contemporaneously determining protein
phosphorylation and phosphorylation changes in toxified
cells. PAO was chosen since it has been shown to be highly
cytotoxic from an earlier study [5], and with the conditions
in this study it had a greater cytotoxic effect than As3+.
Experimental
Reagents
All reagents were of analytical reagent grade. Unless stated
otherwise, all the solutions were prepared in 18 MΩcm−1
doubly deionized water (Sybron Barnstead, Boston, MA,
USA). Tris(hydroxymethyl)aminomethane (Acros Organ-
ics, Morris Plains, NJ, USA) was used as buffer for liquid
chromatography. The buffer pH was adjusted to 7.5 using
hydrochloric acid (Pharmco Products, Inc., Brookfield, CT,
USA). PAO was obtained from Alfa Aesar, Lancaster, UK.
PAO stock solution was prepared in 50% dimethyl
sulfoxide (DMSO; Fisher Scientific, Fairlawn, NJ, USA)
to obtain a stock solution of 200 mmoll−1. The high-
performance liquid chromatography (HPLC) solvents,
water and acetonitrile (ACN), were of high purity and
purchased from Burdick and Jackson (Muskegon, MI).
Sequence-grade-modified trypsin and the acetic acid buffer
were obtained from Promega (Madison, WI). Formic acid
(FA) was purchased from Agilent (USA). Dithiothreitol,
iodoacetamide, protein standards β-casein, bovine serum
albumin (BSA), thyroglobulin, conalbumin, myoglobin,
Arsenic-induced protein phosphorylation changes in HeLa cells

120
and B12 were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Urea and ammonium bicarbonate were from

Cell viability, % of Control

100
Fisher Scientific (Fairlawn, NJ, USA).
80
Cells and culture conditions
60

The HeLa cell lines were available at our labs at University

40
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/
ITMS
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
P
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

Santa Clara, CA, USA) equipped with a binary HPLC
pump, an autosampler, a vacuum degasser system, a
temperature column compartment, and a diode array
detector. The outlet of the UV detector was connected to
sample inlet of the ICP-MS nebulizer using 0.25-mm i.d.
polyether ether ketone tubing of 30 cm in length. For size
exclusion chromatography (SEC), a TSK-GEL G3000SW
(7.5 mm×300 mm, 10 μm) (Tosoh Bioscience LLC, Tokyo,
Japan) was used. The SEC column was calibrated with a
protein mixture of thyroglobulin (670 kDa), conalbumin
(77 kDa), myoglobin (17 kDa), and B12 (1.3 kDa), and they
eluted at 7, 10, 13, and 17 min, respectively. UV absorption
was monitored at 280 nm. The instrumental conditions are
shown in Table 1.
Nano-LC-CHIP/ITMS
All electrospray experiments were done on an Agilent 6300
Series HPLC-CHIP/Ion Trap XCT system (Agilent Tech-
nologies, Santa Clara, CA). The Agilent 1200 LC equipped
with both a capillary and nano-pump was used for loading
and flushing the Agilent chip nanocolumn. The chip used
contained a Zorbax 300SB C18 enrichment column
(4 mm×75 μm, 5 μm) and a Zorbax 300SB C18 analytical
column (150 mm×75 μm, 5 μm). Sample loading onto the
enrichment column was set at a flow rate of 3 μlmin−1 with
a 97:3 ratio of solvent A and B (A 100% H2O, 0.1% FA; B
90% ACN, 10% H2O, 0.1% FA). After loading the
enrichment column, the flush is switched by on-chip
microfluidics to the analytical column at a flow rate of
0.3 μL/min with the following gradient conditions:
0–3 min, 3% B; 3–80 min, 60% B; 80–82 min, 60% B;
82–83 min, 95% B; 83–85 min, 95% B; 85–87 3% B,
followed by 10 min of column re-equilibration. The MS
parameters used for phosphoprotein identification were
shown in Table 1. The target number of ions was 500,000
with a maximum accumulation time of 300 ms. The MS
scan range was 50–2,200m/z in standard enhanced scan
mode. Five parent ions were selected and isolated by the
instrument, producing MS2 and MS3 spectra by collision-
induced dissociation (CID) with He gas and fragmentation
amplitude was set to 1.30 V.
Peptide identification
Database searching was performed on Spectrum Mill
(Rev. A. 03.02) (Agilent Technologies, Santa Clara, CA).
The selected parameters used for the searches were data
searched against the Swiss-Prot database; taxonomy: homo
sapiens; enzyme: trypsin, two missed cleavages; variable
modification: phosphorylation (threonine, serine, tyrosine
or T, S, Y, respectively). The following autovalidation
O. Alp et al.
criteria were used for validation: minimum scores for
spectra resulting from fragmentation of 1+, 2+, 3+, and 4+
for ions were 8, 7, 9, and 9 respectively. Scored peak
intensity (SPI) value was set to at least 70%. Also, in order
to minimize false-positive results, only spectra with a
reversed score that was at least twice smaller than the real
score were taken into account. The data obtained from the
nano-LC-CHIP system were also searched using MASCOT
(Matrix Science, London, UK, online public version).
Swiss-Prot protein database was selected for both search
engines to compare the method confidence in peptide
assignments.
Sample preparation and phosphoprotein fraction
determination by SEC-ICP-MS
PAO was utilized since it produced the maximal toxifica-
tion in the shortest time period, consistent with the project
goals of assessing changes in phosphorylation as a
function of cellular toxicity. PAO treatment was initiated
when the cells were in 80% confluency as estimated by the
microscopic images. Cells were treated with PAO
(0.01–10 μmoll−1) for 3 h and at the end of the incubation
period cells were washed twice with PBS in order to
remove the media residue. After removal of the superna-
tant, the cells were harvested by trypsin–EDTA and then
lysed by M-Per protein extraction buffer (Fisher Scientific,
Fairlawn, NJ, USA). The sample was centrifuged at
13,000×g for 10 min, and the supernatant was taken and
analyzed by SEC-ICP-MS.
Sample preparation for nano-LC-CHIP/ITMS
β-casein and BSA were used as model proteins to ensure
confidence of the method. Tryptic digestion of β-casein and
BSA was performed as follows: 1 mg of β-casein and BSA
was dissolved in 1 ml 50 mM NH4HCO3. A 15-μl portion
of protein mixture was pipetted from the stock solution, and
urea was added to the dissolved protein mixture at a
concentration of 6 M. Then 5 μl of 45 mM dithiothreitol
was added to reduce the protein, and the solution was
incubated at 50 °C for 30 min. After the solution was
cooled to room temperature, 5 μl of 100 mM iodoacetamide
was added, after which, it was left in the dark at room
temperature for 30 min. Sample was diluted with DDI
water in order to have a final concentration of urea less than
1 M. For digestion, 20 μg of trypsin was dissolved in 200
μl of 50 mM acetic acid (as a resuspension buffer). From
this solution, 5 μl of the trypsin solution was added to the
protein mixture and then incubated at 37 °C overnight. To
inhibit the trypsin activity, 3% formic acid was added to the
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)
a
Sum of scores of each identified peptide
b
Scored peak intensity

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

450000
40000
Fraction 4

1 µmol l PAO
-1
400000 35000
(B)
350000 30000
Control
25000
Counts, P
31

300000
(A) 20000
Counts, P

Fraction 3
31

Fraction 2

250000
Fraction 5

15000
Fraction 1

200000 10000

5000
150000
0
100000 7.0 9.3 11.6 13.9 16.2
Time, min
50000

0
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)

1,734.866

1,573.645
1,914.385
1,876.992

1,973.068
2,541.351
2,967.408
2,547.301
2,587.385
1,664.788
2,411.269

inhibiting protein phosphatases or stimulating protein

1,884.93

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.

(M)SKSFQQssLSR(D) (K)IEELDQENEAALENGIK(N)

31
Fraction collection at m/z=31, P

The SEC column effluent was collected as fractions for three

Y947y S948s (R)THTKLSNISESQLDEADFNysRQNGL(-)

(K)QHNsGPNSKPVVSFIAGLTAPPGRR(M)

injections at time intervals that represented the elution of the

(K)tDNTVPFKTPSNEMTPVTIDLVK(K)
(R)FDDGLVHLCWVRSGIsRAALLR(L)
(R)DRIPVySAFRAPRPAPGGAEQR(W)

unidentified phosphorus species some of which are unidenti-

fied phosphorylated proteins. The time intervals for collection
(K)SsSPAPADIAQTVQEDLR(T)

were offset by 0.1 min, to compensate for the lag time from the
(K)HGLSAVGIFtLEySVQR(V)

(K)KDPKQRLGGGsEDAK(E)
(R)LCPQLIIVPPNFDKyR(A)
(K)TQtPPVSPAPQPTEER(L)

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-
Sequence

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

Y261y
Y174y
S231s

S576s

S262s
S396s
T401t

T258t

T370t
Y78y
sites

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
score

50
44

61

34

28

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
14.39
13.61
10.41
10.09
score

scores reported for both search engines are valid scores.

9.63

9.55
9.01
8.77

7.15
8.5

The proteins reported are those which coincide with

10
11

molecular weight ranges associated with the SEC fractions.

1
2
3
4

6
7
8
9
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

(R)LAPITSDPTEATAVGAVEASFK(C) 2,175.118
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
(R)DAGEGLLAVQITDQEGKPK(R) 1,969.024
(K)APLNVQFNSPLPGDAVK(D) 1,766.944
(K)SGCIVNNLAEFTVDPK(D) 1,763.863
(K)VTASGPGLSSYGVPASLPVDFAIDAR(D) 2,547.309
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
with a score of 9.55. This protein is specifically involved in
DNA repair and plays an important role in translesion
synthesis, where the normal high-fidelity DNA polymerases
cannot proceed and DNA synthesis stalls [29]. One of the
results for fraction 2 is the Ras GTPase-activating protein
(GAP), which is a target for protein tyrosine kinases of both
the receptor and cytoplasmic classes and may serve to
integrate tyrosine kinase and Ras signaling pathways [30].
GAP phosphorylated exclusively on serine residues and
recruited to stress granules (SGs) upon either arsenite or
high-temperature treatment [31]. Another important protein
for post-translational modifications is Rho GTPase-
activating protein, identified with a high score of 9.63.
The Rho GTPase-activating proteins are one of the major
classes of regulators of Rho GTPases found in all
eukaryotes that are crucial in cell cytoskeletal organization,
growth, differentiation, neuronal development, and synaptic
functions [32]. Netrin is another PTM-related protein, and
this protein is phosphorylated on cytoplasmic tyrosine
residues and proteolytically cleaved by caspases during
apoptosis [33, 34]. Spectrum Mill results showed two
modifications on tyrosine (Y947y) and serine (S948s).
GC-rich sequence DNA-binding factor protein is involved
in the regulation of transcription in biological process [34].
Matrin 3 is a nuclear matrix protein that has been
implicated in interacting with other nuclear proteins to
anchor hyperedited RNAs to the nuclear matrix. Matrin 3
was identified as both a Ca2+-dependent CaM-binding
protein and a downstream substrate of caspases [35]. Src
substrate cortactin (Amplaxin), which is one of the results
for fraction 3, is related to the reversible protein phosphor-
ylation, and according to the protein search engines results,
Amplaxin has a site modification at T410t. Amplaxin is a
monomeric protein located in the cytoplasm of cells that
can be activated by external stimuli to promote polymeri-
zation and rearrangement of the actin cytoskeleton [36].
Actin is a globular protein found in all eukaryotic cells
which participates in many important cellular processes
such as cell signaling and cell division. The other identified
protein is sphingosine kinase 2 (SPHK2) which catalyzes
the phosphorylation of sphingosine to form sphingosine 1
phosphate. SPHK2 is another member of a growing class of
sphingolipid kinases that may have novel functions such as
apoptosis or inhibition of cellular proliferation [37], which
suggests mediating the cancerous HeLa cell growth. RAC-
alpha serine/threonine protein kinase involves several
different cellular functions, including the control of cell
size and the regulation of survival and metabolism [38].
Two phosphorylated proteins are observed for fraction 4 of
the SEC-ICP-MS chromatogram, and one of them is
endonuclease that has a modification site on Y78y. Its
molecular function is catalysis of the hydrolysis of ester
linkages within nucleic acids and also it can interact non-
covalently with metals and nucleic acids. The other
phosphorylated protein is succinate-CoA ligase. It is a
mitochondrial matrix enzyme and catalyzes the reversible
conversion of succinyl-CoA and ADP or GDP to succinate
and ATP or GTP [39]. Both database search engines did not
give any valid results for fraction 5 as was expected, since
its retention time is around 1 kDa. The results for identified
phosphoproteins are summarized in Table 3. Nevertheless,
the major aim of this study is to identify peptide
phosphorylation sites and possible proteins, and several

proteins are identified at a high confidence level, which are
not phosphorylated but related with post-translational
modifications. The results obtained from Spectrum Mill
and MASCOT are summarized in Table 4. Any of these
proteins, either phosphorylated or PTM-related non-
phosphorylated proteins, have not been observed in the
search results of the control group.
In order to demonstrate the usefulness of the proposed
method, PAO-inoculated total cell lysate was directly
analyzed with nano-LC-CHIP/ITMS system without using
SEC-ICP-MS after tryptic digestion. The results showed
that only a few phosphorylated peptides (sphingosine
kinase 2, Ras GTPase-activating protein, and endonuclease
domain) were matched with the proposed method, and
other phosphorylated proteins could not be identified.
Therefore, to identify phosphorylated proteins using SEC-
ICP-MS monitoring, 31P is needed in order to collect
fractions for preconcentrating and to reduce the sample
complexity before using the nano-LC-CHIP/ITMS system.
Further, in a recent publication by Cvetkovic et al., metal-
loproteins of interest were isolated, utilizing up to six-
dimensional chromatography with ICP-MS detection [40].
Conclusion
The sensitivity of the SEC-ICP-MS in combination with the
nano-LC-CHIP/ITMS is an efficient method to identify
phosphorylated proteins, even in highly complex matrices
such as cell lysate. By using this method, several phosphor-
ylation sites and proteins were identified. Also, obtained
results from Spectrum Mill are compared with MASCOT
protein database search engine, and most of the overlapping
results are at the high confidence level. However, further
studies are necessary to understand the biological relevance
and signal transduction pathways. Understanding these path-
ways should lead to better insight into how the environmental
toxicant arsenic shows its toxic effects regarding the pathol-
ogies associated with arsenic exposure.
Acknowledgements The authors are grateful to Agilent Technologies
for their continuing support via chromatographic and mass spectromet-
ric instrumentation. OA is particularly thankful to the Scientific and
Technical Research Council of Turkey (TUBITAK) for post-doctoral
fellowship support.
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