Total reflection X-ray fluorescence measurements of S ...

Spectrochimica Acta Part B 101 (2014) 118–122

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Total reflection X-ray fluorescence measurements of S and P in proteins using a vacuum chamber specially designed for low Z elements☆ M. Rauwolf a,⁎, C. Vanhoof b, K. Tirez b, E. Maes b, D. Ingerle a, P. Wobrauschek a, C. Streli a a b

Atominstitut, Technische Universität Wien, Stadionallee 2, Vienna 1020, Austria Flemish Institute for Technological Research NV, Boeretang 200, Mol 2400, Belgium

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Article history: Received 15 November 2013 Accepted 17 July 2014 Available online 10 August 2014 Keywords: Total reflection X-ray fluorescence (TXRF) Protein phosphorylation P and S in proteins Low Z elements

a b s t r a c t As the ratio of phosphorus and sulfur in proteins allows the determination of the phosphorylation degree in proteins, the absolute determination of phosphorus and sulfur in organic samples is of growing interest. While it takes some effort to quantify phosphorus and sulfur with inductively coupled quadrupole plasma mass spectrometry (ICP-QMS), total reflection X-ray fluorescence analysis (TXRF) allows easy quantification. In the presented work, the low Z TXRF spectrometer at the Atominstitut was used to analyze phosphorus and sulfur in proteins. Although the preparation of the protein samples proved to be more difficult than originally expected, it could be shown that TXRF is well suited for the determination of P and S in proteins. The obtained lower limits of detection (LLD) for P and S in proteins were extrapolated for 1000s and were 34 pg and 19 pg, respectively. The importance of height scans for each sample to exclude heterogeneities was demonstrated. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Protein phosphorylation is the covalent attachment of phosphate (PO− 4 ) to the amino acid side chains of proteins [1]. This reversible protein phosphorylation regulates cell processes [2] and has significant effects on protein behavior, including enzyme activity and subcellular localization. This post-translational modification is thus an important mechanism in the spatial and temporal regulation of proteins in biological systems. It is estimated that about a third of all eukaryotic cells are phosphorylated at any given time [3]. It is thus not prodigious that abnormal protein phosphorylation is the cause of illnesses such as diabetes and cancer as well as the result of naturally occurring toxins and pathogens [1]. Knowing the extent of phosphorylation in clinical samples can provide a bunch of information as the degree of protein phosphorylation has a direct impact on protein activity. Furthermore, this degree of phosphorylation can be monitored by the phosphorus to sulfur ratio. However, obtaining absolute information regarding the phosphorylation event is challenging, as regular phosphoproteomic approaches only allow relative measurements. The aim of this study was to prove that total reflection X-ray fluorescence analysis (TXRF) can be used to measure P and S in proteins in absolute numbers to gain insight into protein phosphorylation. TXRF is a

☆ Selected paper from the 15th International Conference on Total Reflection X-Ray Fluorescence Analysis and Related Methods, and the 49th Annual Conference on X-Ray Chemical Analysis (TXRF2013), Osaka, Japan, 23–27 Sept. 2013. ⁎ Corresponding author at: TU Wien, Atominstitut, Radiation Physics, Stadionallee 2, 1020 Vienna, Austria. Tel.: +43 1 58801 141339; fax: +43 1 58801 14199. E-mail address: [email protected] (M. Rauwolf).

http://dx.doi.org/10.1016/j.sab.2014.07.022 0584-8547/© 2014 Elsevier B.V. All rights reserved.

special method of energy dispersive X-ray fluorescence analysis (EDXRF) and is characterized by easy sample preparation, small sample volume of a few microliters, simple quantification procedures and excellent detection sensitivities [4–12]. Especially in the area of low Z element analysis, the advantages of TXRF have been demonstrated [13–17]. By using the Atominstitut Low Z TXRF setup, it was possible to measure under vacuum conditions, which allows the detection of P and S at low concentration levels. As the determination of phosphorus and sulfur in biological samples is quite difficult with other analytical methods, such as conventional inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) due to high ionization potentials of P and S and the interferences by polyatomic ions originating from H, C, N and O [18–22], the use of TXRF for this determination is of particular interest. In this study, we applied TXRF measurements using both simple and more complex biological samples to demonstrate the potential of our analytical technique.

2. Experimental 2.1. Samples The liquid samples were prepared at the Flemish Institute for Technological Research NV (VITO) and put on clean round quartz glass reflector plates (30 mm diameter, 3 mm thickness). To prevent spreading of the sample droplets, 5 μl of silicone solution (SERVA in isopropanol) was applied to the reflector plates. The samples were prepared in two groups.

M. Rauwolf et al. / Spectrochimica Acta Part B 101 (2014) 118–122

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Fig. 1. Calibration curves of P and S (Ti as internal standard).

The first series of samples contained HT29 (human Caucasian colon adenocarcinoma) [23], HTC116 (human colon carcinoma) [24] and bis(4-nitrophenyl)hydrogenphosphat (BNPP, C12H9N2O8P) [25] in various concentrations and two blanks. The second set of samples consisted of inorganic P and S samples, BNPP samples in various concentrations and a β-casein sample. For all samples, titanium was used as an internal standard.

2.1.1. Series 1 As complex matrices, two cancer cell lines were used, in which the proteins contained several phosphorylation events. To isolate the proteins, the cells were lysed using 200 μl 50 mM ammonium bicarbonate and sonication (Soniprep 150, MSE, UK). To prevent protein degradation and protein dephosphorylation, both protease inhibitor (1×) and phosphatase inhibitors (3×) were added (Thermo Scientific, Waltham, MA). Of each cell line, one sample was also lysed without the presence of phosphatase inhibitor. After centrifugation (10 min, 4 °C, 13,000 ×g), the supernatants was dialysed for 4 hours at 4 °C. Next, the protein concentration was determined using the Qubit Protein assay kit (Life Technologies, Carlsbad, CA). Of each sample, 100 μl was mixed with 100 ppm Ti as an internal standard. Subsequently, 10 μl of each sample was pipetted in the centre of a round quartz reflector plate. The reflector plate was put on a heating plate (approximately 70 °C) for 3 minutes to allow drying of the sample.

Fig. 2. Atominstitut's low Z TXRF setup.

XRF reflector plates. After drying, the reflector plates were ready for measurement. 2.2. Calibration The calibration used to quantify the series of measurements was set up at the Atominstitut using aqueous ICP standards (Merck, CertiPUR) showing perfect linearity between counts and concentration (Fig. 1). Ti was used as internal standard. To test the calibration Seronorm™ Trace Elements Serum L-2 (SERO AS, Billingstad, Norway) and BNPP samples with different P and S concentrations were measured and evaluated and quantified using QXAS-AXIL [26]. Each sample was prepared

2.1.2. Series 2 Non-complex phosphorus (and sometimes sulfur) containing solutions including BNPP, inorganic phosphorus and beta-casein (which is a protein containing 5 phosphorylation sites), were used to prepare samples of known concentrations. In 90 μl of each sample, 10 μl 100 ppm Ti standard was added before spotting 10 μl sample onto

Table 1 Calibration check with seronorm trace elements serum L-2. P values are certified. S values are non-certified. Expected concentration values determined after dilution with ultrapure water. Seronorm™ Trace Elements Serum L-2 Element

Expected concentration (ppm)

Measured concentration (ppm)

P S P S

8.1–10.2 129.7 3.0–3.8 48.53

10.4 ± 0.4 130.1 ± 6.7 3.87 ± 0.01 52.8 ± 1.1

Fig. 3. Spectra of samples 2 and 7 (first series of measurements). Spectrum of HT29-1 was rescaled to fit the Ti peak of the BNPP sample.

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Table 2 Results of the first series of measurements. Expected concentrations calculated after sample preparation. No.

Sample

Expected P concentration (μg/ml) ⁎

1 2 3 4 5 6 7 8 9 10 11 12 13

Blank HT29-1 HT29-2 HCT116-2 HCT116-3 BNPP 10 μg/ml BNPP 1 μg/ml BNPP 0.1 μg/ml HT29-1 HT29-2 HCT116-2 HCT116-3 Blank

– 0.892 1.08 1 1.2 10 1 0.1 0.892 1.08 1 1.2 –

Measured P concentration (μg/ml) 0 203.8 257.3 223 ± 0.1 175.1 7.9 0.9 0.1 206.2 316.6 187.1 176.3 0

± 3.3 ± 14.9 ± ± ± ± ± ± ± ±

10.0 0.1 0.04 0.02 1.0 7.3 8.2 3.2

Measured S concentration (μg/ml) 0 138.7 194.2 113.1 118 ± 2.2 0.3 0 0 137.9 175.7 115.8 111.3 0

± 0.8 ± 6.6 ± 0.6 ± 0.02

± ± ± ±

2.8 15.9 2.3 0.4

⁎ For the cell cultures, the amount of phosphorus present was estimated as the obtained protein concentration times 0.04%.

twice on two different reflectors. The quantification of the BNPP samples resulted in the measured P concentrations 94.7 ± 1.6 ppm and 49.6 ± 5.6 ppm for expected concentrations of 91.1 ppm and 50.7 ppm, respectively. As can be seen in Table 1, the quantification of the Seronorm™ Trace Elements Serum L-2 samples also provided satisfying results.

2.3. Analytical procedure The measurements were performed at the low Z TXRF vacuum chamber spectrometer of Atominstitut [14], which is shown in Fig. 2.

As the X-ray fluorescence energies of low Z elements are in the range of 1 keV and their intensities are several magnitudes lower compared to medium Z elements, it is very important to reduce all kinds of absorbers between sample and active volume of the detector. Absorption in air can be prevented by measuring under vacuum conditions. Due to the sample preparation, a thin sample film can be assumed. Therefore, for the quantification, no sample self-absorption has to be taken into account and the thin film approximation can be used. A detector with an ultrathin window (Moxtek AP3.3, 300 nm polymer on Si grid support) is required to reduce absorption of the low energy radiation in the detector window. Optimum excitation conditions are achieved by the use of a Cr anode tube [14]. The X-ray tube was operated at 30 kV and 30 mA and

Fig. 4. Height scan for sample 5 (inorganic P + S) with spectra obtained at the respective height values.


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Table 3 Results of the second series of measurements. Expected concentrations calculated after sample preparation. No.

Sample

1 2 3 4 5 6 7 8 9 10 11 12 13

Inorganic P Inorganic P Inorganic P Inorganic P Inorganic P Inorganic P BNPP BNPP BNPP BNPP BNPP BNPP β-Casein

+ + + + + +

S S S S S S

Expected P concentration (μg/ml)

Expected S concentration (μg/ml)

0.1 1 10 10 100 1 1 50 0.1 1 100 10 20

0.1 1 10 10 100 1 – – – – – – 20

had an optical focus 40 μm × 8 mm. For these measurements, a 30 mm2 Oxford Premium grade B35 detector (with an UTW and a FWHM at of 134 eV at 5.9 keV [15]) and a XIA Saturn Digital X-ray processor were used. Monochromatic Cr Kα radiation selected from the full spectra by a Ni/C multilayer (2d = 12 nm) was used to perform the measurements. Each sample was measured for 1000 s. 2.4. Measuring protocol An aliquot of 10 μl of the sample was pipetted on 30 mm diameter quartz reflectors, which were previously checked for cleanness. The measuring protocol consisted of a height scan of the sample (as the sample is mounted on a translation stage horizontally) to check the optimal sample position referring to the exciting beam: the sample is moved through the exciting beam to bring the sample and the footprint of the beam below the detector. This is a standard procedure for TXRF adjustments. For samples of similar thickness, we expected to find the intensity maxima for Ti (and all other elements in the sample) at the same height position. If the element distribution within the sample is homogeneous, the position of the maximum of the fluorescence intensity is found at the same height for all elements in the sample. All samples were measured twice for 1000s each. For the second measurement of each sample, the quartz reflector was turned about 90° to 180°. The comparison of those two measurements was used to gain an insight into the homogeneity of the element distribution in the sample. If the quantification of those two measurements does not result in the same concentrations for P and S, we found evidence of a heterogeneous sample distribution. The arithmetic mean of the P and S concentrations of these two measurements for each sample was used to evaluate the samples.

Measured P concentration (μg/ml) 0 0.6 ± 6.0 ± 5.1 ± 22.1–71 0.6 ± 1.2 ± 36.0 ± 0.4 ± 0.8 ± 63.2 ± 7.5 ± 8.6 ±

0.01 0.7 0.9 0.02 0.1 0.4 0.02 0.4 6.8 1.5 0.1

Measured S concentration (μg/ml) 0 0.9 ± 0.03 5.5 ± 0.7 5.6 ± 0.1 15.9–85.8 0.9 ± 0.01 0 0 0.5 ± 0.1 0 0 0 11.8 ± 0.1

two spectra, the spectrum of HT29-1 was rescaled to fit the Ti peak of the BNPP sample. The comparison of those two spectra shows that the P and S concentrations in sample 2 were a lot higher than expected. For the cell cultures, the amount of phosphorus present was estimated as the obtained protein concentration times 0.04% (= theoretical P abundance). Generally, it turned out that the concentrations obtained for the protein samples HTC116 and HT29 were about a factor 200 higher than the expected concentrations (Table 2) and that no differences were seen between the presence and absence of phosphorylation inhibitors. The reason was found in the presence of huge amounts of P and S in the protease inhibitor and phosphatase stability buffers used for sample preparation. Besides the throughout dialysis, P and S were still present in the sample; although in smaller amounts than in the original sample. Even buffer exchange protocols were not able to reduce the amount of P and S to a background level (data not shown). Nonetheless, a good detection limit for P (65 pg) was obtained from the measurements for BNPP (sample 7) using the measured P concentration. 3.2. Second series of measurements The distribution of P in the samples appeared to be heterogeneous in sample 4 (deviation of more than 30% between the P values, while the deviation of the S values was below 5%), 10 (more than 65% deviation) and 12 (more than 30% deviation). In sample 5 (inorganic P + S) Ti was distributed heterogeneously (Fig. 4.), while P and S appeared to be distributed homogeneously. Therefore, the exact concentrations of P and S in sample 5 (Table 3) could not be determined. Results range between 22 and 71 μg/ml and 16 and 86 μg/ml, respectively. Using the measured concentrations from sample 4 (inorganic P + S) the detection limits for P (34 pg) and S (19 pg) for the low Z TXRF were calculated. 4. Conclusion and outlook

3. Results and discussion The quantification of protein phosphorylation in biological samples is of tremendous importance in many clinical applications. To determine the total phosphorus content in these samples, we demonstrated that TXRF applications can be applied. In this study, we used both standard inorganic solutions and single proteins, as well as very complex cell culture samples to test this analytical platform. 3.1. First series of measurements The distribution of elements in the samples of the first series of measurements was homogeneous as no significant deviations between the two measurements of the same sample have been obtained. Fig. 3 shows a spectrum for sample 2 (HT29-1) and sample 7 (BNPP containing 1 μg/ml P). Both samples contained 10 μg/ml Ti. To reduce the high detector dead time due to the higher concentrations in the sample (HT29-1), sample 2 was measured at a smaller angle. To compare the

The sample preparation turned out to be more complicated than originally expected. Nonetheless, it could be shown that TXRF is well suited to analyze phosphorus and sulfur in proteins and thus has a possibility to be included in phosphorus quantification in biological samples. Some samples showed heterogeneous element distributions when measuring height scans and different points on the sample. Based on the sufficient detection limits for P (34 pg) and S (19 pg), we propose that TXRF is a suitable method to analyze protein fractions. This would reveal an insight into the positions of the phosphate groups in the protein. This pilot study shows that the TXRF technique can complement more regular phosphoproteomics techniques. For example, when complex samples are separated in less-complex protein fractions using liquid chromatography, TXRF can be used to quantify the absolute phosphorus content in these fractions, and in this way, looking for phosphorus content differences between different samples.

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