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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- 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


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


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, 79].

and the cytotoxicity of arsenic degradation products is due to differences in cellular response [ 5

O. Alp et al.

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

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 ( μ LC) 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 contemporane ously 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 As 3+ .

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 major ity 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

All reagents were of analytical reagent grade. Unless stated otherwise, all the solutions were prepared in 18 M Ω cm 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,

iodoacetamide, protein standards β - casein, bovine serum albumin (BSA), thyroglobulin, conalbumin, myoglobin,

Arsenic-induced protein phosphorylation changes in HeLa cells

and B 12 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Urea and ammonium bicarbonate were from 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-cm 2 flasks and maintained in Dulbecco s Modified Eagle s Medium (DMEM: Fisher Scientific, Fairlawn, NJ, USA) supple- mented with 10% fetal bovine serum (Fisher Scientific, Fairlawn, NJ, USA) at 37 °C with 5% CO 2 in a humidified atmosphere. Subculture passages were performed every 3 days using trypsin ethylenediaminetetraacetic acid (EDTA): 0.05% trypsin 0.53 mM EDTA×4Na (Gibco Invitrogen Corporation, Carlsbad, CA, USA). All PAO solutions were made up fresh by diluting the stock solution in DMEM media prior to dosing. PAO was added to cell culture flasks at the indicated times and concentrations below prior to harvest. After treating the cells with PAO, mitochondrial function was measured using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of cell viability.

Cytotoxicity assay (MTT assay)

HeLa cells were seeded in 24-well plates at a density of 1×10 4 ml 1 and incubated for 24 h before adding various concentrations of PAO (0.01, 0.1, 1, 5, and 10 μ moll 1 ). Cells were exposed to PAO at indicated concentrations for 3, 6, and 24 h. A working solution of MTT reagent was dissolved in 1-ml phosphate saline buffer (PBS; Gibco Invitrogen Corporation, Carlsbad, CA, USA) at a concentration of 5 mgml 1 and diluted to a volume of 50 ml with media. After removing the media in each well at the end of exposure time, 1 ml of MTTmedia solution was placed in each well and incubated for 3 h. After removing the media MTT solution, 0.5 ml DMSO was added to each well. Then the culture plate was placed on a micro-plate reader and the absorbance measured at 550 nm with background correc- tion. Results are shown in Fig. 1 .


Inductively coupled plasma mass spectrometry

An Agilent 7500ce ICP-MS (Agilent Technologies, Santa Clara, CA, USA) was used for phosphorus ( 31 P) detection. It is equipped with an octopole cell and can be operated with or without the collision/reaction gas. Helium (He) was used as collision gas at a flow rate of 3.6 mlmin 1 throughout the experiments. A conventional Meinhard

120 100 80 60 40 20 0 0.01 0.1 1 5 10 Cell viability, %
Cell viability, % of Control

PAO concentration, µmol l -1

viability, % of Control PAO concentration, µmol l - 1 3 hours 6 hours 24 hours

3 hours

% of Control PAO concentration, µmol l - 1 3 hours 6 hours 24 hours Fig.

6 hours

of Control PAO concentration, µmol l - 1 3 hours 6 hours 24 hours Fig. 1

24 hours

Fig. 1 Dose-dependent viability changes in PAO-exposed HeLa cells. Data are shown as percentages of the control value. Each value represents the average ± SD of quadruplicate wells

nebulizer, a Peltier-cooled spray chamber (2 °C), and a shield torch constitute the sample introduction system under standard plasma conditions. Instrument conditions are given in Table 1 .

High-performance liquid chromatography

Chromatographic separations were carried out with an Agilent 1100 liquid chromatograph (Agilent Technologies,

Table 1 Operating conditions for ICP-MS, HPLC, and nano-LC-CHIP/ ITMS

ICP-MS parameters

Forward power, W


Plasma gas flow rate, lmin 1


Carrier gas flow rate, lmin 1


Isotope monitored

31 P

Collision gas (He) flow rate, mlmin 1


Quadrupole bias, V


Octopole bias, V


Energy discrimination voltage


SEC-HPLC parameters



Mobile phase

30 mM Tris HCl buffer, pH 7.5

Flow rate, mlmin 1


Injection volume, μ l


Nano-LC-CHIP/ITMS parameters

Drying gas, N 2 ,

4 lmin -1



MS capillary voltage

1,900 V


30 V

Capillary exit

128 V

Trap drive

75 V

300°C MS capillary voltage 1,900 V Skimmer 30 V Capillary exit 128 V Trap drive 75

O. Alp et al.

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 μ m) (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 B 12 (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 .


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 μ m, 5 μ m) and a Zorbax 300SB C18 analytical column (150 mm×75 μ m, 5 μ m). Sample loading onto the enrichment column was set at a flow rate of 3 μ lmin 1 with a 97:3 ratio of solvent A and B (A 100% H 2 O, 0.1% FA; B 90% ACN, 10% H 2 O, 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 μ L/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 2,200 m /z in standard enhanced scan mode. Five parent ions were selected and isolated by the instrument, producing MS 2 and MS 3 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

or T, S, Y, respectively). The following autovalidation criteria were used for validation: minimum scores for

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 μ moll 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 NH 4 HCO 3 . A 15- μ l 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 μ l 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 μ l 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 μ g of trypsin was dissolved in 200 μ l of 50 mM acetic acid (as a resuspension buffer). From this solution, 5 μ l 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


# of matched peptide



MW, Da Protein name


# of matched peptide


MW, Da Protein name

score a


score a

1 1




β -casein (bovine)




β -casein (bovine)

2 12




Serum albumin (bovine) 19



Serum albumin (bovine)

a Sum of scores of each identified peptide b Scored peak intensity

were passed through 0.22- μ m 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 μ moll 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 μ moll 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 μ moll 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 400000 1 µmol l -1 PAO 35000 (B) 30000 350000 Control 25000 300000
1 µmol l -1 PAO
Time, min
Counts, 31 P
Counts, 31 P
Fraction 1
Fraction 2
Fraction 3
Fraction 4
Fraction 5

Time, min

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

moll − 1 PAO exposure time 3 h (measured at m / z 3 1 P),

O. Alp et al.

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

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

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

Src substrate cortactin (Amplaxin) (SEC fraction 3)

Ras GTPase-activating protein (SEC fraction 2)

Rho GTPase-activating protein (SEC fraction2)

DNA polymerase kappa (SEC fraction 1)

Netrin receptor UNC5D (SEC fraction 2)

Endonuclease domain (SEC fraction 4)

Sphingosine kinase 2 (SEC fraction 3)

Matrin-3 (SEC fraction 2)

Matched parent Protein name mass (Da)













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












Spectrum Mill MASCOT Variable

S8s S9s




































8.77 9.55 7.15 9.63 9.01 8.5 – 9 10 11 3 4 5 6 1 2

species possibly because the cell membrane may be more permeable to As species with phenyl groups [ 5 ]. Since the amount of phosphorylated proteins was expected to be very limited, 3 h of PAO exposure time was chosen in order to assure 70% viable cells for SEC- ICP-MS experiments.

SEC-ICP-MS results

HeLa cells were incubated for 3 h with various concen- trations of PAO (0.01 10 μ moll -1 ). When SEC-ICP-MS chromatogram of 1 μ moll 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 PAO-inoculated group are in the range of 1 100 kDa. On the other hand, with up to 0.1 μ moll 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 inhibiting protein phosphat ases or stimulating protein kinases, since these enzymes, respectively, are related with dephosphorylation and phosphorylation events in cells.

Fraction collection at m /z =31, 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 NH 4 HCO 3 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

Protein identification of each fraction was performed by 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



Matched parent

Protein name



mass (Da)

1 19.28




Pyruvate kinase isozymes M1/M2




2 19.09




Clathrin heavy chain 1

3 16.16




Serine/threonine-protein phosphatase PP1

4 15.75




60S ribosomal protein L19

5 14.54





6 14.43














7 13.56




Stress-70 protein

8 12.22




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 Ca 2+ -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 prote in 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

Nevertheless, the major aim of this study is to identify peptide phosphorylation sites and possible proteins,

O. Alp et al.

proteins are identified at a high confidence level, which are not phosphorylated but relate d 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, 31 P 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].


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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