Investigations of Sulfur Chemical Status with ...

Send Orders for Reprints to [email protected] Protein & Peptide Letters, 2016, 23, 000-000

1

Investigations of Sulfur Chemical Status with Synchrotron Micro Focused X-ray fluorescence and X-ray Absorption Spectroscopy Hiram1 A. Castillo-Michel1*, Angel G. Diaz-Sanchez2, Alejandro Martinez-Martinez2 and Bernhard Hesse 1

European Synchrotron Radiation Facility, B.P. 220 Grenoble, France; 2Instituto de Ciencias Biomedicas, Universidad Autónoma de Ciudad Juárez, Anillo envolvente Pronaf, 32310 Cd. Juárez, México Please provide Abstract: Sulfur (S) is an essential macronutrient for all living organisms. A variety of organic and corresponding author(s) photograph inorganic S species with oxidation states ranging from -2 to +6 exist. Today few spectroscopic and size should be 4" x 4" inches biochemical methods are used to investigate sulfur oxidation state and reactivity in biological samples. X-ray absorption near edge spectroscopy (XANES) is a very well suited spectroscopic technique to probe the oxidation state and the surrounding chemical environment of sulfur. Microspectroscopy beamlines, operating at almost all synchrotron facilities, allow the combination of XANES with X-ray fluorescence mapping (XRF). Using this approach distribution maps of S in complex biological samples (intact parts of tissue, or individual cells) can be obtained using XRF and its oxidation state can be probed in-situ (XANES). Moreover, XRF mapping at specific energies enables for chemical contrast of S at different oxidation states without the need of staining chemicals. This review introduces the basic concepts of synchrotron XRF and XANES and discusses the most recent applications in life science. Important methodological and technical issues will be discussed and results obtained in different complex biological samples will be presented.

Keywords: Oxidation state, chemical imaging, elemental mapping, core level spectroscopy, cysteine, cystine, radiation damage. IMPORTANCE OF SULFUR CHEMICAL STATUS Sulfur (S) is an essential macronutrient for all living organisms, a variety of organic and inorganic S species exist with oxidation states ranging from -2 to +6. The most abundant form of S present in biomolecules and macromolecules is as thiol (R-SH) from cysteine (Cys) residues. The Cys amino acid is present in glutathione, a compound essential in the response to oxidation damage in cells [1]. It is also essential for the synthesis and function of proteins [1]. The wide number of oxidation state modifications that can occur in the thiol group, allow this residue to perform a countless number of biochemical and physiological functions in living organisms. Oxidation of the thiol group in Cys leads to formation of sulfenic (Cys-SO), sulfinic (Cys-SO2) and sulfonic (CysSO3) groups, the last one considered as an irreversible state [2]. The oxidation of S in Cys residues is essential for the formation of covalent cross-links, named disulfide bridges that stabilize the three-dimensional functional structure of many proteins in cells. Thiol groups from Cys residues function as strong nucleophiles in several enzymatic active sites, the thiolate state being the most reactive one. With this respect the oxidation state of S in Cys residues found in macromolecules is hypothesized to be an important point of regulation of protein function. Oxidizing conditions in cells induce shifts of S oxidative state in Cys, sulfenic and sulfinic *Address correspondence to this author at the European Synchrotron Radiation Facility, B.P. 220 Grenoble, France; Tel: 33(6)4 76 88 29 48; Fax: 33(6) 476 88 27 85; E-mail: [email protected] 0929-8665/16 $58.00+.00

derivates are formed in a reversible manner, thus oxidationreduction of Cys appears to be an important cell redox signaling molecular switch (see Fig. 1). It is well known that the thiol group chemistry, which is structurally and functionally relevant to protein and peptide function, must be taken into account in complex biological studies. Today few spectroscopic and biochemical methods are used to investigate S oxidation state and reactivity in biological samples (Table 1). X-ray absorption near edge spectroscopy (XANES) is a very well suited spectroscopic technique to probe oxidation state and surrounding chemical environment of S. The energy of the absorption edge for S at different oxidation states spans over about 13 eV (see Fig. 2 as an example of S XANES at different oxidation states) [3]. The energies at which transitions are observed provide specific information about the bonding character of the S atom. Moreover, with the use of a microfocused beams distribution maps of S in complex biological samples (intact parts of tissue, or cell cultures) can be obtained using -Xray fluorescence (XRF) and its oxidation state can be probed in-situ (XANES). Synchrotron XRF and XANES X-ray absorption near edge spectroscopy (XANES) is a technique that exploits the features in the absorption spectra originating from the photo absorption cross section of electronic transitions from an atomic core level to final states in the 50-100eV energy region above the ionization energy of the core level [3]. It is an element-specific spectroscopic © 2016 Bentham Science Publishers

2 Protein & Peptide Letters, 2016, Vol. 23, No. 3

Castillo-Michel et al.

Figure 1. Oxidation reactions at the S atom of protein Cys residues. (1) thiol group deprotonation generating the thiolate group, a more reactive nucleophile; (2) thiolate group can suffer a series of oxidations generating sulfenic; (3) sulfinic and (4) sulfonic acids; (5) Thiolate nucleophilic attack to a substrate, generating a productive protein-substrate complex; (6) in some proteins thiolates react with oxidized glutathione forming a mixed disulfide; (7) that can react with another cysteine and generate a disulfide bond (Cystine); (8) disulfides can be oxidized to generate thiosulfinate and (9) subsequently thisulfonate; finally (10) the irreversible oxidized forms of Cys residues lead to protein degradation. Most enzymes that use Cys residues for catalysis use nucleophilic addition on substrate groups thus performing covalent catalysis (R–X). Table 1.

Analytical methods commonly used to investigate sulfur chemical status. Method

Target S species

Immunochemical

Sulfenic

Requirements Preparation of specific antibodies

Reference [4]

Sample treatment with dimedone In vitro Immunodetection system X-ray Crystallography

NMR

sulfenic, sulfinic, sulfonic, and disulfide sulfenic

Highly purified protein

[5-9]

Protein crystals X-Ray diffractometer Highly purified protein

[10, 11]

Labeled protein NMR spectrometer Mass spectrometry

UV-absorbance

Benzylamine

sulfenic, sulfinic and sulfonic


Thiol, thiolate, and disulfide


Sulfenic

C14 labeled benzylamine

[8, 12-14]

Mass spectrometer [15, 16]

Protein mutagenesis in some cases, chemical treatment with DTT or other reductor [17]

Fast determination due to sulfonamides instability 5-mercapto-2-nitrobenzoic acid

Sulfenic

Anaerobic conditions Protein denaturation and trypsin treatment

[18, 19]

Chromatographic detection of Cys peptide derivative 7-chloro-4-nitrobenz-2-oxa-1,3-diazole

Sulfenic

Indirect methods

Sulfenic, disulfide and others

Protein purification and chemical modification Protein denaturation and free thiol chemical protection. Subsequent reduction of sulfenic state cysteine and quantification

[20] [21-25]

Investigations of Sulfur Chemical Status with Synchrotron Micro Focused

technique that provides information about the chemical state and local geometry of the absorbing element. Moreover, no long-range ordering of the investigated material is required and hence XANES allows for the investigation of materials in all states of matter. Semi quantitative evaluation of the data is possible using linear combination fitting to known references or by modeling the resonances with Gaussian curves [26]. Since this technique requires an intense tunable X-ray source it is almost exclusively offered at synchrotron facilities. XANES spectra acquisition can be done in transmission and fluorescence mode where the incident energy is selected and scanned with the use of double crystal monochromators (typically Si crystals for selecting energies above 1.9 keV). In transmission the signal is obtained by monitoring the beam intensity before and after the sample with the use of ionization chambers or diodes. In fluorescence mode, the intensity of an emission line associated to the absorption process is monitored using an energy dispersive detector (most often solid state detectors made of Silicon or Germanium). The fluorescence mode is preferred for diluted samples and due to its multi-elemental detection capacity is used on imaging applications to obtain elemental distribution maps in heterogenous samples. Detection limits for S in fluorescence mode are in the order of M to mM (or femtograms to nanograms with a 1m2 beam) when exciting at energies close to the K binding energy (as usually done for XRF/ XANES applications). Microspectroscopy beamlines, operating at almost all synchrotron facilities, allow the combination of XANES with X-ray fluorescence mapping (XRF) by means of micro and nano beams (a non-comprehensive list can be found in [27]). A recent general lay out and description of the typical components of a microspectroscopy beamline can be found

Protein & Peptide Letters, 2016, Vol. 23, No. 3

3

in [28] and [29]. Due to the high absorption of X-rays at energies close to the S k-edge beamlines typically operate under vacuum or include a chamber to flow a light inert gas (e.g. Helium). Combining XRF and XANES is a very powerful strategy that consists of raster scanning a sample at fixed incident energy and collecting a fluorescence spectrum at each point (pixel) to obtain elemental distribution maps after plotting the integrated count rate in a specific region of interest of the XRF spectrum or after fitting all spectra [30]. From the XRF images, points of interest are selected to perform XANES (see Fig. 3). The points of interest are usually selected based on co-localization of elements and/or intensity of the element of interest. However, this point by point XANES acquisition is often not enough to provide the full detail of the chemical species present in heterogeneous samples; the full 2D speciation is needed to be able to reach sound conclusions. In recent years, different set-ups have been proposed to extend XANES punctual acquisitions to full 2D. In transmission mode, a series of X-radiographies of the samples are acquired, while scanning the energy of the incoming “large” beam across the edge of the element of interest. This fullfield XANES (FF-XANES) method has been successfully applied in the study of cultural heritage specimens, fuel cells, batteries and catalysts [31-33] (see [33] and [34] for more details about this technique). However, its applicability to the life sciences is restricted due to the typically low S concentration in the samples that yield low local absorption in transmission. In fluorescence mode, XRF maps can be taken at few specific energies ( 2-5) where contrast from chemical species/oxidation states can be obtained by calcu-

Figure 2. XANES spectra of S compounds with different oxidation states. Spectra were obtained from CaSO42H2O (sulfate), Cysteic acid (sulfonic), Cysteine (thiol), and Cystine (disulfide) all analyzed as solid finely ground powder. Cysteine reference spectrum and cysteic acid were obtained from ID21 reference library (http://www.esrf.eu/UsersAndScience/Experiments/Imaging/ID21/php/allbase).



Figure 3. Typical combined XRF/XANES analysis of a lymphocyte cell. A) XRF distribution map of S obtained with 3KeV incident beam (1m2 pixel size). B) XRF spectrum of the full map showing the fit for different elements performed using PyMCA [30]. The blue shadowed-box corresponds to the region of interest used to obtain the XRF intensity for S XANES. C) XANES spectrum obtained from the spot marked on A.

lating ratios to obtain chemical speciation maps [35, 36]. However, this method does not provide a full XANES spectrum and can be difficult to distinguish species with similar XANES signal (e.g. Cysteine from methionine). In recent years, the so-called “full spectrum XANES” (FS-XANES) XRF mapping has emerged as a novel approach to acquire a XANES spectrum from each pixel of an image by scanning the same sample area at multiple (~ few hundred) energies along an element’s absorption edge. This has been possible thanks to the development of new XRF detectors [37, 38] (e.g. applications in environmental science and cultural heritage [39] [40] [41]). FS-XANES is indeed the appropriate choice to study S distribution and speciation in biological samples. However, the disadvantage of this mode of analysis is the long acquisition times and potential radiation damage. At this moment, performing S k-edge FS-XANES on biological samples is challenging due to the high dose delivered that may lead to unreliable XANES spectra. For this reason, XRF chemical contrast is more commonly used to obtain 2D speciation data. However, the acquisition time for XRF imaging has been decreasing significantly due to advances in XRF detector electronics and pulse processing algorithms [37]. The use of intense micro-focused beams makes the sample more prone to radiation damage. Since the XANES spectrum is a weighted sum of the species present in

the illuminated area of the sample, there is a threshold at which the appearance of new species due to radiation damage has an impact on the XANES spectrum. The key point when acquiring XANES is to be able to minimize radiation damage such that the spectrum remains unaffected. In point by point XANES the acquisition of fast scans or on the fly energy scans (acquisition time of few seconds to minutes) are used as strategy to minimize radiation damage. Upgrades of present SR sources and new facilities under construction will produce more brilliant and coherent X-ray beams [42]. Hence, fast acquisition scanning setups will be needed to perform XRF/XANES analyses at higher spatial resolutions in larger areas and keep samples stable under these very intense micro and nano beams. Here, we describe the more recent applications of combined XRF and XANES for investigating S chemical state in biological samples. SULFUR XRF AND XANES APPLICATIONS IN LIFE SCIENCES The use of synchrotron XRF/XANES to localize and chemically probe S oxidation state has been demonstrated in several studies in the life sciences. A review of recent literature is presented herein not only emphasizing the scientific results but also important methodological and technical as-


pects (e.g. sample preparation, radiation damage issues). This revision is limited to applications that have taken advantage of the combination of S mapping (XRF) and oxidation state in-situ probing (XANES). Hence, reports on the use of S bulk XANES and pure S mapping are not within the scope of this review. Human Brain Tissue The distribution of S in neuromelanin containing neurons from human brain tissue and its oxidative state have been studied by means of XRF/XANES [43]. The experiment performed on 8m thick tissue sections from formalin-fixed, paraffin embedded human brain tissues from young and aged individuals provided new information regarding the S environment in neuromelanin. The main contributions observed in the XANES spectra were identified as i) thiols (-SH) and monosulfides (R-S-R’) such as glutathione and methionine, ii) sulfoxides such as methionine sulfoxide, and iii) sulfonate species such as cysteic acid and taurine. The comparison between age groups showed that during the first years of life S in neuromelanin is mainly present in the reduced form (organic monosulfide/thiols) with low proportions of oxidized forms (sulfoxide/sulfonate). During childhood and adolescence sulfonate contribution increases while thiols and organic monosulfide contributions are decreased. Finally at advanced age organic monosulfide and sulfonate species dominate with low contributions from thiols and sulfoxide. The presence of the highly oxidized S forms (sulfoxide and sulfonate) in neuromelanin is suggestive of oxidative degradation of the pigment. This study was complimented with hard X-ray high-spatial resolution XRF to investigate the presence of other elements such as Fe and Cu in neuromelanin. The results showed for the first time the presence of structures in the pigment able to bind metals. The authors suggest that those metal-binding domains are physiologically active structures that are part of an adaptation process to efficiently bind potentially toxic metals. This report also addressed sample preparation and radiation damage critical issues regarding XRF/XANES analysis in bran tissues. The authors acknowledged that formalin fixation and paraffin embedding may induce changes on the samples, including changes in elemental concentrations. To account for this all samples were carefully prepared following the exact same protocol to minimize inconsistent artifacts in all tissues and make them comparable. As reported in [44] formaldehyde fixation in rat cerebellum tissue sections induces a loss of content of sulfonic acids. This is attributed to the loss of taurine during immersion in the aqueous formaldehyde solution. Furthermore, they report a reduction in the total sulfur content and the highest relative concentration of disulfides and sulfoxides in formalin-fixed tissue. Hence, the recommendation for tissue sample preparation is unfixed frozen hydrated specimen analyzed under cryo-conditions. However, these kinds of samples are much more difficult to obtain and pose particular constraints regarding transport and experimentation. Alternatively, freeze drying unfixed tissue is most likely to preserve the S redox state of the tissue and help prevent radiation damage [44]. Radiation damage was assessed thanks to the use of rapid continuous XANES scans (100ms dwell time per energy point). Consecutive scans from the same point were compared and radiation damage


5

was assessed looking at the evolution of a peak at around 2470eV which is attributed to inorganic sulfides produced by photoreduction of oxidized sulfur species (sulfonate and sulfoxide). The chemical forms of S in glial brain tumors were investigated using point by point XANES and XRF chemical contrast imaging [36]. The punctual XANES acquisition revealed the exclusive presence of S in the -2 oxidation state, but no specific attribution of the signal to organic monosulfide or disulfide was performed. Using the XRF chemical contrast imaging approach the authors investigated S oxidative stress distribution and noticed the presence of discrete regions containing oxidized S. This approach consisted on the acquisition of XRF maps at specific energies 2473.5, 2476.4, 2482.5 and 2500eV which allow selective excitation of S-2, S+4 (plus S-2), S+6 (plus S-2 and S+4) and total sulfur, respectively. In order to obtain the correct 2D chemical state maps it was necessary to subtract the contributions from lower oxidation states calculating their ratios. An important fact addressed in this study is that performing XRF mapping at multiple energies induces shifts in the beam position making the calculation of ratios inaccurate. To avoid this, images have to be registered or aligned so that the subtraction gives the correct result. The authors propose a routine to for image alignment, other alignment methods are implemented in the software PyMCA [30] [45]. Another important issue is the consideration of self-absorption in XRF detection techniques. Self-absorption is the loss of XRF intensity due to the inherent properties of the sample, is dependent on the mass attenuation coefficient of the sample for S emission lines and sample thickness. The authors estimated less than 4% loss of intensity for their brain tissue samples (4m thickness) and hence this was considered negligible. However, this needs to be considered more seriously for thicker samples (15m and above). Corneal Animal Tissue Another application of S k-edge μXRF chemical contrast imaging is a study of the differential S speciation in bovine corneal tissue [46]. To achieve corneal transparency the collagen fibrils have to be lattice-like arranged. This is, in part, controlled by sulfated proteoglycans, which, via core proteins, bind to the collagen at specific locations along the fibril axis. Data reported in [46] yielded the relative percentage of each sulfur species in the tissue and reports an inhomogeneity in the composition of S species in the first 50 μm of stromal depth. In addition, they disclose the spatial distribution of S species (thiol/organic monosulfide and sulfate) and the co-localization of phosphorous with thiols/organic monosulfides in the epithelial region of corneal tissue (Fig. 4). The authors point out the advantage of synchrotron XRF ‘label free’ chemical imaging with respect to histo- and immunochemical methods since it provides the unaltered S biochemical signature. Plant Tissue Synchrotron techniques in plant science are becoming more and more often used for investigating chemical status of both nutrient and toxic elements [47]. Due to its important biological role in plant biology, S chemical status has been



Figure 4. Sulfur speciation in corneal tissue. Using the chemical contrast provided by tuning the X-ray beam energy to enhance the absorption from sulfate (top map) and thiol/organic monosulfide (bottom map) (with permission from [46]).

often investigated by means of XANES (as reviewed in [3]). However, despite of the numerous cases where S has been identified as the ligand for toxic metals such as Hg, Cd, Ag, Cu, As, and Pb [47], investigations with combined XRF and XANES at the S k-edge are scarce. More often S is mapped simultaneously with other elements using XRF to confirm co-localization with elements such as the abovementioned. Besides applications towards understanding detoxification of toxic metals, investigating S chemical status in plants can provide confirmation of some metabolic routes as it has been reported in [48]. Onion (Allium cepa) fully hydrated tissue sections were investigated using XRF chemical contrast to elucidate the distribution of organic disulfide, organic monosulfide, sulfoxide and sulfate. S chemical species were first investigated by bulk XANES before and after inducing cell breakage in the tissues. Cell breakage exposes the molecular precursor S(1-propenyl) cysteine sulfoxide present in cytoplasm to the enzyme alliinase which is preferentially located in bundle sheath cell. Upon cell breakage the sulfoxide molecule is converted to propensulfenic acid which is turn converted to the lachrymatory factor (propanethial S-oxide) responsible of the well known lachrymatory properties of onions. Results reported in [48] confirm the above mentioned chemical pathway, XANES spectra revealed a decrease in the amount of sulfoxide groups in onion tissue after cell breakage and the presence of lachrymatory factor and thiosulfinate S species. The XRF chemical contrast approach revealed the predominant localization of sulfoxide groups in the periphery of cortex cells, in agreement with the hypothesized cytosolic compartmentalization of the sulfoxide precursor in onion. Investigations in red onions revealed similar distribution and content of sulfoxide and sulphide/thiol but in contrast to green onion disulfides are also prominent particularly in the transport bundle region. This is indicative of a more oxidized environment in the transport vessels. Cytosolic compartmentalization of the sulfoxide precursor was also observed in the red onions. This investigation demonstrated the application of S XRF chemical contrast imaging in living plant tissues. No radiation damage issues were reported in this work for the imaging approach; however in the full XANES collections were restricted to only one scan as subsequent scans were accumulating chemical changes from photo-oxidation products.

Biomineralization in Mollusk Shells XRF and XANES are considered essential techniques aiding to our better understanding of biomineralization processes [28]. The minerals of biogenic origin are well known to differ from their nonbiogenic equivalents. The role of S in the biomineralization process has been extensively studied in calcareous skeletons ([28] [49] and references there in). Models suggest the secretion of a mineralizing sulfated organic rich matrix is the first step in the process that is followed by the crystallization phase in which mineral material grows into the organic framework. XRF chemical imaging at the S k-edge is an unchallenged method to map the distribution of organic matrices (usually containing SH/S-S groups) in calcareous biominerals. As an example in the brachiopod Terebatulina retusa, XRF maps depicted the distribution of sulfate across the primary and secondary layers of the dorsal valve [50]. The primary region is more sulfate rich compared to the secondary region, whereas S in the form of thiol and/or disulfides is localized in the sheaths surrounding the calcite fibers and is co-localized with phosphorus (Fig. 5). To address the question whether the sulfate in shells of T. retusa is organic or inorganic single point XANES spectra were acquired and evidenced the contribution of organic sulfate. Radiation Damage In order to illustrate the effect of radiation damage on biological samples a set of experiments were performed at beamline ID21 of the European Synchrotron Radiation Facility [51]. The beam size was defined using a 100m pinhole for experiments with low photon density (unfocused) resulting in a photon flux of 2.07 x 105 photons/s/m2. For high photon density experiments, conditions used for imaging applications, the beam was focused with the use of a set of fixed curvature KB mirrors (0.8x0.3m2) providing a flux of 5.71x1010 photons/s/m2. XANES spectra were acquired from 2450-2550eV with 0.3eV energy steps and 100ms dwell time per energy point using the standard set-up of ID21 as reported in [46]. XANES analyses were performed to investigate the well known fast degradation of disulfide bonds under X-ray beams [52]. For this purpose, recom-


Figure 5. Sulfur speciation maps in primary and secondary layer of T. retusa dorsal valve depicting the distribution of (a) sulfate, (b) thiol/disulfide and (c) phosphorus (with permission from [50]).


7

Figure 6. XANES spectra of protein DJ1 obtained with focused and unfocused. Linear combination fit obtained using CaSO4 (sulfate), Cysteic acid (sulfonic) and Cystine (disulfide). Data analysis done using the linear combination fit module of ATHENA software [54].

binant DJ-1 obtained from E. coli BL21(DE3) (Agilent) and blood circulating lymphocyte cells extracted from RAT species as reported in [53] were used. XANES spectra of protein DJ1 obtained with focused and unfocused beam are shown in Fig 6. Linear combination fitting of DJ1 unfocused XANES gave a composition of 93% sulfonic, 6% disulfide and 3.2%. Radiation damage products appeared as new features on the XANES spectrum attributed to inorganic sulfides and sulfoxide (see Fig 6). For the lymphocyte sample, linear combination fitting of XANES from lymphocyte cells gave a composition of 81% thiol, 12.3% disulfide, 3.5% sulfonic and 3.2% sulfate (see Fig. 7). After 20 scans with unfocused beam no radiation damage effects were observed, but the presence of a shoulder at 2471.05eV characteristic of inorganic sulfides was observed after one scan with focused beam. As expected radiation damage effects on biological samples during S k-edge XANES acquisition are not negligible with microfocused beams. In order to prevent these effects, analyses should be performed with lower photon densities (e.g. below a total dose of 6.2x106 photons/m2 per scan), detection should be enhanced by using more sensitive and larger solid angle fluorescence detectors, and the use of cryogenic sample environment is highly recommended. Radiation damage must be carefully studied for each sample and for particular beamline configurations; however the values reported here provide a valuable reference for beamlines based on undulator synchrotron sources with KB focusing optics. The results favored the strategy of performing XRF chemical contrast imaging as it is presented in [36] and [46]. XRF chemical imaging allows extracting oxidation state information in 2D from radiation sensitive samples by

Figure 7. XANES spectra of Lymphocytes obtained with focused and unfocused beam. Linear combination fit obtained using CaSO4 (sulfate), Cysteic acid (sulfonic) and Cystine (disulfide). Data analysis done using the linear combination fit module of ATHENA software [54].

reducing the acquisition dwell time (and as direct result the radiation dose) at individual spots from 60s (1 full energy scan) to 1s (for the case of maps at 5 different energies with 200ms dwell time per pixel).



CONCLUSIONS

[9]

Synchrotron radiation-based methods are currently being used as an approach for acquiring unique structural details of biological samples. Novel synchrotron radiation methods and application of those that already exist are essential to provide vital mechanistic and biological information. The use of XRF and XANES seems to be a well suited path for mapping changes in the S oxidation state of biological samples (e.g. cells, tissues and protein preparations) under stress conditions such as disease, infections, or other conditions that alter the oxidative state of living organisms. Careful sample preparation protocols must be followed to obtain meaningful results. Whenever possible unfixed frozen hydrated state samples will preserve conditions close to in vivo state. Unfixed freeze dried is then a second option that avoids artifacts induced by fixative chemicals. Radiation damage effects on biological samples are not negligible under intense microfocused beams. In all cases the best compromise between flux and detection should be found to avoid exposing the sample to unnecessary photon doses. In that sense, to these days, XRF chemical contrast imaging seems to be the only feasible acquisition mode to assess S chemical status in 2D for biological samples.

[10] [11]

[12] [13]

[14] [15]

[16]

[17]

CONFLICT OF INTEREST The authors state no conflict of interest. Financial support for the writing of this manuscript was provided by the European Synchrotron Radiation Facility and Universidad Autónoma de Ciudad Juarez.

[18] [19]

ACKNOWLEDGEMENTS All authors have contributed significantly to the writing of this manuscript. We acknowledge the European Synchrotron Radiation Facility and beamline ID21 for providing beamtime. We acknowledge Mariana Grigoruta, Alejandra Vargas and Emmanuel Vazquez from Universidad Autónoma de Ciudad Juarez for providing samples for XANES analysis presented in this manuscript.

[20]

[21] [22]

REFERENCES [1] [2] [3] [4] [5]

[6] [7] [8]

Jocelyn, P.C. Biochemistry of the SH group, Academic Press Inc: 1972. Montfort, R.L.; Congreve, M.; Tisi, D.; Carr, R.; Jhoti, H. Oxidation state of the actvie-site cysteine in protein tyrosine phosphatase 1B. Nature, 2015, 423, 773-777. Jalilehvand, F. Sulfur: not a "slient" element any more. Chem. Soc. Rev., 2006, 35, 1256-1268. Miseta, A.; Csutora, P. Relationship between the ocurrence of cysteine in proteins and the complexity of organisms. Mol. Biol. Evol.,2000, 17, 1232-1239. Pe'er, I.; Felder, C.E.; Man, O.; Silman, I.; Sussman, J.L.; Beckman, J.S. Proteomic signatures: amino acids and oligopeptide compositions differentiate among phyla proteins. Struc. Funct. Bioinform., 2004, 54, 20-40. Stehle, T.; Claiborne, A.; Schulz, G.E. NADH binding site and catalysis of NADH peroxidase. Eur. J. Biochem, 1993, 211, 221226. Yeh, J.I.; Claiborne, A.; Hol, W.G. Structure of the native cysteinesulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8A resolution. Biochem., 1996, 35, 9951-9957. Poor, C.B.; Chen, P.R.; Duquid, E.; Rice, P.A.; He, C. Crystal structures of the reduced, sulfenic acid, and mixed disulfide forms of SarZ, a redox active global regulator in Staphylococcus aureus. J. Biol. Chem., 2009, 284, 23517-23524.

[23] [24] [25] [26]

[27]

[28] [29]

Dombkowski, A.A., Sultana, K.Z., Craig, D.B. Protein disulfide engineering. FEBS lett., 2014, 588, 206-212. Crane, E.J.; Vervoort, J.; Claiborne. A. 13C NMR analysis of the cysteine-sulfenic acid redox center of enterococcal NADH peroxidase. Biochem., 1997, 36, 8611-8618. Nakamura, T.; Yamamoto, T.; Manabu, A.; Matsumura, H.; Hagihara, Y.; Goto, T.; Yamaguchi, T.; Inoue, T. Oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate. PNAS, 2008, 105, 6238-6242. Fuangthong, M.; Helmann, J.D. The OhrR repressor senses organic hydroperoxides by reversible formation of cysteine-sulfenic acid derivative. PNAS, 2002, 99, 6690-6695. Boschi-Muller, S.; Azza, S.; Sanglier-Cianferani, S.; Talfournier, F.; Van Dorsselear, A.; Branlat, G. A sulfenic acid enzyme intermediate is involved in teh catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. J. Bio. Chem., 2000, 275, 35908-35913. Kaiserova, K.; Srivastava, S.; Hoetker, J.D.; Awe, S.O.; Tang, X.L.; Cai, J.; Bhatnagar, A. Redox activation of aldose reductase in the ischemic heart. J. Biol Chem., 2006, 281, 15110-15120. Benitez, L.; Allison, W.S. The inactivation of the acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3phosphate dehydrogenase by dimedone and olefins. J. Biol. Chem, 1974, 249, 6234-6243. Vignaud C., Rakotozafy, L., Falquieres, A., Potus, J., Nicolas, J. Separation and identification by gel filtration and hihg-performance liquid chromatography with UV or electrochemical detection of the disulphides produced from cysteine and glutathione oxidation. J. Chromatogr.,2004, 1031, 125-133. Allison, W.S., Benitez, L.V., Johnson, C.L. The formation of a protein sulfenamide during the inactivation of the acyl phosphatase activity of oxidized glyceraldehide 3-phosphate dehydrogenase by benzylamine. Biochem. Biophys. Res. Commun.,1973, 52, 14031409. Poole, L.; Claiborne, A. The non-flavin redox center of the streptococcal NADH peroxidase II. Evidence for a sstabilized cysteine-sulfenic acid. J. Biol. Chem, 1989, 264, 12330-12338. Ellis, H.R.; Poole, L.B. Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium. Biochem., 1997, 36, 13349-13356. Ellis, H.R.; Poole, L.B. Novel application of 7-chloro-4nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide. Biochem., 1997, 36, 15013-15018. Eaton, P. Protein thiol oxidation in health and disease: techniques for measuring disulfides and related modifications in complex protein mixtures. Free Radic. Bio. Med., 2006, 40, 1889-1899. Ishii, T.; Sunami, O.; Nakajima, H.; Nishio, H.; Takeuchi, T.; Hata, F. Critical role of sulfenic acid formation of thiols in teh inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide. Biochem. Pharmacol.,1999, 58, 133-143. Jaffrey, S.; Solomon, H.S. The biotin switch method for the detection of S-nitrosylated proteins. Sci. Signaling, 2001, 86, 11. Saurin, A.; Neubert, H.; Brennan, J.P.; Eaton, P. Widespread sulfenic acid formation in tissues in response to hydrogen peroxide. PNAS, 2004, 101, 17982-17987. Karisch, R.; Neel, B.G. Methods to monitor classical proteintyrosine phosphatase oxidation. FEBS J., 2013, 280, 459-475. Prietzel, J.; Botzaki, A.; Tyufekchieva, N.; Bretthole, M.; Thieme, J.; Klysubun, W. Sulfur speciation in soil by S k-edge XANES spectroscopy: Comparison of spectral deconvolution and linear combination fitting. Env. Sci. Technol., 2011, 45, 2878-2886. Majumdar, S.; Peralta-Videa, J.R.; Castillo-Michel, H.; Hong, J.; Rico, C.M.; Gardea-Torresdey, J.L. Applications of synchrotron microXRF to study the distribution of biologically important elements in different environmental matrices: A review. Anal. Chim. Acta, 2012, 755, 1-16. Dauphin, Y.; Salome, M. In: Characterization of biominerals and biomimetic materials; Laurie B. Gower Ed.; CRC Press: Boca Raton, 2014, pp. 73-93. Pushie, "Elemental and chemically specific X-ray fluorescence imaging of biological systems," Chemical reviews, vol. 114, pp. 8499-8541, 2014.

Investigations of Sulfur Chemical Status with Synchrotron Micro Focused [30] [31]

[32]

[33] [34]

[35] [36]

[37]

[38]

[39]

[40]

[41]

Solé, V.A.; Papillo, E.; Cotte, M.; Walter, Ph.; Susini, J. A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra. Spectrochim. Acta part B, 2007, 62, 63-68. Meirer, F.; Liu, Y.; Pouyet, E.; Fayard, B.; Cotte, M.; Sanchez, C.; Andrews, J.; Mehta, A.; Sciau, P. Full-field XANES analysis of Roman ceramics to estimate firing conditions- A novel probe to study hierarchical firing conditions- Anovel probe to study hierarchical heterogenous materials. J. Anal. At. Spectrom., 2013, 28, 1870-1883. Pouyet, E.; Cotte, M.; Fayard, B.; Salome, M.; Meirer, F.; Mehta, A.; Uffelman, E.S.; "2D X-ray and FTIR micro-analysis of the degradation of cadmium yellow pigment in paintings of Henri Matisse," Appl. Phys. A, Vols. DOI 10.1007/s00339-015-9239-4, 2015. Andrews, J.C.; Weckhuysen, B.M. Hard-Xray spectroscopic Nanoimaging of hierarchical functional materials at work. Chem. Phys. Chem., 2013, 14, 3655-3666. Fayard, B.; Pouyet, E.; Berruyer, G.; Bugnazet, D.; Cornu, C.; Cotte, M.; De Andrade, V.; Di Chiaro, F.; Hignette, O.; Kieffer, J.; Martin, T.; Papillon, E.; Salome, V.A. The new ID21 XANES fullfield end-station at ESRF. J. Physics: Conf. series, 2013, 425, 192001. Marcus, M. X-ray photon-in/photon-out methods for chemical imaging. TrAC, 2010,508-517. Szcerbowska-Boruchowska, M.; Stegowski, Z.; Lankosz, M.; Szpak, M.; Adamek, D. A synchrotron radiation micro-X-ray absorption near edge structure study of sulfur speciation in human brain tumors- a methodological approach. J. Anal. At. Spectrom., 2012, 27, 239-247. Siddons, D.; Kirkham, R.; Ryan, C.G.;De Geronimo, G.; Dragone, A.; Kuczewski, A.J.; Li, Z.Y., Carini, G.A.; Pinelli, D.; Beuttenmuller, R.; Elliot, D.; Pfeffer, M.; Tyson, T.A.; Moorhead, G.F.; Dunn P.A. Maia X-ray microrprobe detector array system. J. of Physics: Conf. Series,2014, 499, 012001. Tack, P.; Garrevoet, J.; Bauters, S.; Vekemans, B.; Laforce, B.; Van Ranst, E.; Banerjee, D.; Longo, A.; Bras, W., Vincze, L. Fullfield fluorescence mode micro-XANES imaging using a unique energy dispersive CCD detector. Anal. Chem., 2014, 86, 87918797. Etschman, B.; Donner, E.; Brugger, J.; Howard, D.; de Jonge, M., Paterson, D., Naidu, R., Scheckel, K., Ryna, C., Lombi, E. Speciation mapping of environmental samples using XANES imaging. Environ. Chem., 2014, 11, 341-350. Monico, L.; Janssens, K.; Alfeld, M.; Cotte, M.; Vanmeert, F.; Ryan, C.; Falkenberg, G.; Howard, D.; Brunetti, B. Full spectral XANEs imaging using the Maia detector array as a new tool for the study of the alteration process of chrome yellow pigments in paintings by Vincent van gogh. J. Anal. At. Spectrom. , 2015, 30, 613-626. Keune, K.; Mass, J.; Meirer, F.; Pottasch, C.; van Loon, A.; Hull, A.; Church, J.; Pouyet, E.; Cotte, M.; Mehta, A. Tracking the transformation and transport of arsenic sulfide pigments in paints: synchrotron based X-ray micro-analyses. J. Anal. At. Spectrom., 2015, 30, 813-827.

Received: October 21, 2015

Revised: December 16, 2015

Accepted: December 16, 2015

Protein & Peptide Letters, 2016, Vol. 23, No. 3 [42] [43]

[44]

[45] [46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

9

Reich, E.S. Ultimate upgrade for US synchrotron. Nature, 2013, 501, 148-149. Bohic, S.; Murphy, K.; Werner,P.; Cloetens, P.; Salome, M.; Susini, J.; Double, K. Intracellular chemical imaging of the developmental phases of human neuromelanin using synchrotron X-ray microspectroscopy. Anal. Chem., 2008, 80, 9557-9566. Hackett, M.; Smith, S.E.; Paterson, P.G.; Nichol, H.; Pickering, I.; George, G. X-ray absorption spectroscopy at the sulfur K-edge: A new tool to investigate the biochemical mechanisms of nuerodegeneration. ACS Chem. Neurosci., 2012, 3, 178-185. Paleo, P., Pouyet, E., Kieffer, J. Image stack alignment in full-field X-ray absorption spectroscopy using SIFT_pyOCL. J. Synch. Rad., 2014, 21, 456-461. Veronesi, G.; Koudouna, E.; Cotte, M.; Martin, F.L., Quantock, A.J. X-ray absorption near-edge structure (XANES) spectroscopy identifies diffrential sulfur speciation in corneal tissue. Anal. Bioanal. Chem, 2013, 405, 6613-6620. Sarret, G., Pilon-Smits, E.A.H., Castillo-Michel, H., Isaure, M.P., Zhao. F.J., Tappero, R. Use of synchrotron-based techniques to elucidate metal uptake and metabolism in plants," in Advances in Agronomy, Donal Sparks Ed.; Academic press Elsevier, 2013, pp. 1-82. Pickering, I., Sneeden, E.Y., Prince, R.C., Block, E., Harris, H.H., Hirsch, G., George, G.N. Localizing the chemical forms of sulfur in vivo using X-ray fluorescence spectroscopic imaging: Application to onion (Allium cepa) tissues. Biochem., 2009, 48, 6846-6853. Cuif, J.P.; Dauphin, Y.; Nehrke, G.; Nouet, J.; Perez-Huerta, A. Layered growth and crystalization in calcareous biominerals: Impact of structural and chemical evidence on two major concepts in invertebrate biomineralization studies. Minerals,2012, 2, 11-39. Cusack, M., Dauphin, Y., Cuif, J.P., Salome, M., Freer, A., Yin, H. Micro-XANES mappinf of sulphur and its association with magnesium and phosphorus in te shell of the brachiopod Terebratulina retusa. Chem. Geol.,2008, 253, 3-4. Salome,M.; Cotte, M.; Baker, R.; Barret, R.; Benseny-Cases, N.; Berruyer, G.; Bugnazet, D.; Castillo-Michel, H.; Cornu, C.; Fayard, B.; Gagliardini, E.; Hino, R.; Morse, J.; Papillon, E.; Pouyet, E.; Rivard, C.; Sole, V.A.; Susini, J.; Veronesi, G. The ID21 Scanning X-ray Microscope at ESRF. J. Phys.: Conf. Ser. , 2013, 425, 182004. Sutton, K.A.; Black, P.J.; Mercer, K.R.; Garman, E.F.; Owen, R.L.; Snell, E.H.; Bernhard, W. Insights into the mechanismis of X-ray induced disulfide-bond cleavage in lysozyme crystals based on PER, optical absorption and X-ray diffraction studies. Acta Cryst , 2013, 69, 2381-2394. Vargas-Caraveo, A.; Castillo-Michel, H.A.; Mejia-Carmona, G.E.; Perez-Ishiwara, G.; Cotte, M., Martinez-Martinez, A. Preliminary studies of the effects of psychological stress on circulating lymphocytes analyzed by synchrotron radiation based-Fourier transform infrared microspectroscopy. Spectrochim. Acta A Mol. Biomol. Spectrosc., 2014, 128, 141-146. Ravel, B.; Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Rad., 2005, 12, 537-541.