Analytical Biochemistry 499 (2016) 85e89
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A strategy to quantitate global phosphorylation of bone matrix proteins _ Grazyna E. Sroga, Deepak Vashishth* Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2015 Received in revised form 7 January 2016 Accepted 25 January 2016 Available online 3 February 2016
Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured “in bulk” are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials. © 2016 Elsevier Inc. All rights reserved.
Keywords: Global phosphorylation Human bone Mouse bone Extracellular matrix proteins Osteopontin
Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and the landscapes of their phosphorylation in regulation of different cellular functions. Mass spectrometry (MS)1 has become the technique of choice to identify phosphorylated residues within proteins, whereas fragmentation analysis by tandem mass spectrometry (MS/MS) is used to confirm the peptide sequence and to assign the site of modification [1e4]. It is worth noting that the reliable localization of phosphorylation sites in proteins represents one of the most challenging tasks in phosphoproteomic studies. Although powerful, MS tends to be biased toward certain phosphorylated sites, and in general it is not quantitative. Thus, routine analyses of, for example, age-related changes in global phosphorylation levels of proteins
* Corresponding author. E-mail address: [email protected] (D. Vashishth). 1 Abbreviations used: MS, mass spectrometry; MS/MS, tandem mass spectrometry; WT, wild-type; OPN ( / ), osteopontin knockout; CK2, casein kinase II; rhOPN, recombinant human osteopontin; MT plate, microtiter plate; avidineHRP, avidin-linked horseradish peroxidase; PTM, post-translational modification. http://dx.doi.org/10.1016/j.ab.2016.01.017 0003-2697/© 2016 Elsevier Inc. All rights reserved.
using MS equipment are impractical for daily tests performed on patient specimens in a present-day clinical setting. The quantitative measurement of global protein phosphorylation in comparison with the total amount of protein is essential to clinical and pharmacological research and to biomedical applications. From the perspective of bone tissue research, such studies are particularly important for, for example, the understanding of biomineralization regulation occurring in vivo [5] and/or biomineralization performed in vitro for various purposes of bone tissue engineering [6]. Thus, for the needs of bone research and biomedical engineering, we developed a strategy that combines in vitro phosphorylation and dephosphorylation of mineralized bone with an assay for the quantification of global phosphorylation of bone matrix proteins. The phosphorylation levels were measured “in bulk” using a pIMAGO-based system recently developed by Iliuk and coworkers [7]. In the current study, we have, for the first time, quantified global phosphorylation levels of extracellular bone matrix proteins isolated from bone tissues. The samples originated from male and female human cadaveric donors of different ages as well as from wild-type (WT) and osteopontin knockout [OPN ( / )] C57BL/6 mice. To this end, we were able to capture a
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G.E. Sroga, D. Vashishth / Analytical Biochemistry 499 (2016) 85e89
difference in the global phosphorylation levels between matrix proteins originating from WT and OPN ( / ) mice. We also demonstrated that there is a difference in the levels of global phosphorylation between bone matrix proteins isolated from human cortical and trabecular bone. We expect that the developed strategy has potential to open new avenues for studies on global phosphorylation of bone matrix proteins and their role in bone biomineralization and fragility fractures as well as for other tissues/cells and protein-based materials. Materials and methods Human and mouse bone samples Tibias (posterior area) from a total of 8 human female (4) and male (4) cadaveric donors (young [25.0 ± 2.0 years], middle age [61.0 ± 2.0 years], and elderly [85.0 ± 2.0 years] donors as well as 1 female donor of 77 years) served as the source of cortical and/or trabecular (spatially matched) bone tissue samples. The specimens were obtained from the centralized National Disease Research Interchange (NDRI) biobank and were known to be free of osteoarthritis, diabetes, and other metabolic bone diseases as well as HIV and HVB. Tibias from a total of 3 WT and 3 OPN ( / ) C57BL/6 mice were harvested, cleaned, and cut using a slow-speed diamond blade (Buehler, Lake Bluff, IL, USA). The mice bones served as the source of cortical bone. Both human and mice bone pieces were repeatedly washed in cold distilled water until the washings were free of blood and then were defatted using isopropyl ether. After freeze-drying, the specimens were stored at 80 C until their use. Dephosphorylation and phosphorylation of mineralized bone samples and osteopontin Three types of experiments were performed on bone samples and osteopontin (recombinant human osteopontin, R&D Systems, Minneapolis, MN, USA): phosphorylation, dephosphorylation, and non-enzyme-treated control reactions. Phosphorylation was conducted by incubating the samples overnight at 30 C with casein kinase II (CK2) and the reaction buffer from New England Biolabs (Ipswich, MA, USA). Adenosine triphosphate was added to the buffer as the phosphoryl donor for CK2. The incubation solution also contained 50 mM CaCl2, protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1, Pierce Biotechnology, Rockford, IL, USA) and antibiotics (ampicillin and kanamycin). Dephosphorylation was performed by incubating the samples overnight at 37 C with calf intestinal alkaline phosphatase and the reaction buffer from New England Biolabs. The incubation solution also contained 50 mM CaCl2, protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1) and antibiotics (ampicillin and kanamycin). All control reactions contained only the enzyme incubation buffers supplemented with 50 mM CaCl2, protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1), and antibiotics (ampicillin and kanamycin). Isolation of bone matrix proteins for pIMAGO-based assay Extracellular bone matrix proteins were isolated as described by Sroga and coworkers [8,9] with some modifications to the published procedures. Specifically, in addition to protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1), a mixture of phosphatase inhibitors (100 Halt Phosphatase
Inhibitor Cocktail, final concentration 1, Pierce Biotechnology) was added to the protein extraction buffer. Quantitation of global phosphorylation levels for extracellular bone matrix proteins and osteopontin using pIMAGO-based assay Quantitative measurement of global phosphorylation levels in protein extracts isolated from each bone sample and all control reactions was performed as described by Iliuk and coworkers [7] using a newly developed pIMAGO phosphoprotein detection system. Briefly, the extracted proteins, recombinant human osteopontin (rhOPN), and phosphorylated casein (additional phosphoprotein control [7]) were bound to the wells of the polystyrene microtiter plates (MT plates). The PBS buffer alone (0.5 and 1) and plasmid DNA alone (0, 10, 20, 40, 80, and 100 ng DNA/ well) were also included in the assay as controls. After a series of blocking (Tris-buffered saline with 5% nonfat dried milk, pH 7.4) and washings (Tris-buffered saline with 0.1% Tween 20, pH 7.4) steps, the pIMAGO reagent (Tymora Analytical, West Lafayette, IN, USA) was added to the MT plate wells to allow the attachment of pIMAGO to the phosphate groups on proteins. The pIMAGO is a water-soluble nanopolymer that is functionalized with titanium(IV) ions that specifically bind to phosphoproteins. The nanopolymer also contains biotin groups that allow the enzyme-linked spectrophotometric detection. In the next step, pIMAGO excess was removed by a minimum of three washings (Tris-buffered saline with 0.1% Tween 20, pH 7.4), and the avidin-linked horseradish peroxidase (avidineHRP) was added to the wells in order to proceed with the colorimetric detection. The absorbance of the solutions in each well was determined at 415 nm using a microtiter plate reader (Infinite 200, Tecan). Results and discussion In vitro dephosphorylation and phosphorylation of mineralized bone samples It was necessary to modify the composition of the buffers supplied with enzymes from New England Biolabs for the needs of the reactions performed on mineralized bone tissues. Thus, to maintain bone's mineral content, 50 mM CaCl2 was used. Preservation of the natural mineral content in bone samples is important if, for example, bone fracture tests were going to follow the phosphorylation/dephosphorylation procedures. To block the activity of proteases, which are naturally present in the extracellular bone matrix, a cocktail of protease inhibitors was added to the reaction buffers. In addition to sterilization of the used solutions (i.e., depending on the compounds, autoclaving or sterile filtration), antibiotics (ampicillin and kanamycin) were also added to prevent the potential growth of bacteria. Isolation of bone matrix proteins for pIMAGO-based assay For the analysis of global protein phosphorylation, protein extracts should reflect as accurately as possible the natural phosphorylation levels of all proteins isolated, for example, at a certain age of a donor or at a particular time point from implanting a graft. Thus, to prevent undesired dephosphorylation of bone matrix proteins after the completion of all the reactions, the cocktail of phosphatase inhibitors was also introduced to the protein extraction buffer in order to block the activity of alkaline phosphatases naturally present in bone matrix.
G.E. Sroga, D. Vashishth / Analytical Biochemistry 499 (2016) 85e89
Specific and highly sensitive measurement of global phosphorylation levels of extracellular bone matrix proteins Before quantifying natural global phosphorylation levels of bone matrix proteins isolated from different human and mice bones, we tested the specificity and sensitivity of the pIMAGO nanopolymer on a smaller set of samples, which included the in vitro phosphorylated and dephosphorylated rhOPN and cortical bone samples originating from the 77-year-old Caucasian female donor. After isolation of extracellular matrix proteins from the bone samples, the respective protein extracts and phosphorylated/dephosphorylated rhOPN samples were bound to the wells of the MT plates. In addition, different concentrations of plasmid DNA alone (0, 10, 20, 40, 80, and 100 ng DNA/well) and PBS buffer alone (0.5 and 1) were bound to the wells of the MT plates (Fig. 1). They served as independent controls of the pIMAGO nanopolymer specificity. Using these additional controls, we demonstrated that the pIMAGO nanopolymer does not bind to the free phosphate groups or DNA. Notably, our protein extraction procedure [8,9] was developed to ensure that there is no detectable DNA or hydroxyapatite in the prepared protein extracts. Still, it was important to show that the aforementioned compounds do not interfere with the determination of the global phosphorylation levels of the extracellular bone matrix proteins when the pIMAGO nanopolymer is used. We were particularly interested in osteopontin because it not only is one of the major phosphoproteins of bone matrix but also is significantly phosphorylated in bone [4,10]. In addition, OPN is extensively altered through other types of post-translational modifications (PTMs) such as glycosylation and proteolytic processing. All PTMs of OPN have significant effects on the structure and biological properties of the protein [11,12]. The nature of OPN phosphorylation is highly tissue and cell specific, reflecting the diverse functions of the protein in different physiological environments. Human OPN has 36 identified phosphorylation sites [13]. The 36 amino acid residues that undergo phosphorylation represent the maximal level of OPN modification because significant variability referring to those 36 phosphorylation sites was observed within and between different cells and tissues [13,14]. Therefore,
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we assumed that due to both the high level of OPN phosphorylation and its high matrix content, even small changes in the global OPN phosphorylation would be less complicated to follow as compared with other less abundant and not as extensively phosphorylated proteins. We demonstrated that under the developed pIMAGO-based assay conditions, the level of global phosphorylation of rhOPN determined before the in vitro phosphorylation was approximately 0.532 ± 0.012 A415/100 ng rhOPN and increased to approximately 1.326 ± 0.004 A415/100 ng rhOPN after the treatment using CK2 (Fig. 2A). Thus, we confirmed that we do have capabilities to alter and measure global phosphorylation levels of a protein. Notably, during our experiments phosphorylation and dephosphorylation of rhOPN was conducted to completion. Therefore, the resulting phosphorylation pattern of rhOPN was most likely different from the in vivo phosphorylation patterns observed for bone matrix OPN [5]. The ability to control and quantify the levels of global phosphorylation of OPN (as well as other phosphoproteins of bone matrix known as SIBLING proteins [12]) is of great interest for the field of bone tissue engineering [4,5] because this phosphoprotein was demonstrated to regulate hydroxyapatite mineralization [14,15]. Next, we showed that the in vitro phosphorylation and dephosphorylation of matrix proteins in human cortical bone of tibia from the 77-year-old Caucasian female indeed changed the natural (0.616 ± 0.004 A415/100 ng of protein extract) levels of phosphorylation (Fig. 2B). Thus, after the in vitro phosphorylation, the global phosphorylation levels of the extracellular bone matrix proteins isolated from three cortical bone pieces of the posterior area were similar and comprised 0.993 ± 0.010 A415/100 ng of protein extract. The in vitro dephosphorylation led to stripping of phosphate groups from the matrix proteins to nearly the same level of 0.228 ± 0.019 A415/100 ng of protein extract. Hence, we established that it is possible to modulate in vitro the levels of bone matrix phosphorylation, an issue that is essential to the field of bone tissue engineering. Moreover, we embarked on the quantitation of global bone matrix phosphorylation in WT and OPN ( / ) knockout mice. We hypothesized that due to the lack of OPN in the OPN ( / ) mice, one could observe a difference in the global phosphorylation level of extracellular matrix proteins isolated from bones of WT and OPN ( / ) mice (Fig. 2C and D). Indeed, we determined that the natural phosphorylation levels were slightly higher in the WT mice bones (0.612 ± 0.010 A415/100 ng of protein extract) when compared with the OPN ( / ) mice bones (0.474 ± 0.015 A415/100 ng of protein extract). Similarly, after the in vitro dephosphorylation, the levels of phosphorylation declined to 0.330 ± 0.010 A415/100 ng of protein extract. Hence, we demonstrated that the pIMAGO-based assay is sensitive enough to detect differences in the global phosphorylation levels between OPN and all other proteins of the extracellular bone matrix. Global phosphorylation levels of extracellular matrix proteins differ between human cortical and trabecular bone
Fig.1. Photograph of an MT plate with the results of the pIMAGO-based assay. The following samples were adsorbed into the wells of the plate. Controls: b-casein (100 ng/well, row A, lanes 1e3), 0.5 PBS buffer (row A, lanes 4e6), 1 PBS (row A, lanes 7e9). Protein extracts from 77-year-old Caucasian female bone samples: dephosphorylated (rows BeD, lanes 1e3), control: native phosphorylation (rows EeG, lanes 1e3), phosphorylated in vitro (rows BeD, lanes 4e6). Recombinant human OPN in triplicate (20, 50, and 100 ng/well): dephosphorylated (rows EeG, lanes 4e6), control: original phosphorylation (rows BeD, lanes 7e9), phosphorylated in vitro (rows EeG, lanes 7e9). DNA in duplicate (0, 10, 20, 40, 80, and 100 ng DNA/well) (rows BeG, lanes 10 and 11).
The developed strategy was ultimately used for the measurement of natural levels of the global protein phosphorylation in the extracellular matrix proteins isolated from the spatially matched cortical and trabecular bone samples isolated from tibias (posterior area) of human female and male cadaveric donors of three age groups (young [25.0 ± 2.0 years], middle age [61.0 ± 2.0 years], and elderly [85.0 ± 2.0 years] donors) (Fig. 3). We established that the natural levels of human cortical bone phosphorylation measured “in bulk” were on average 0.818 ± 0.17 A415/100 ng of protein extract for male donors (Fig. 3A) and 0.663 ± 0.07 ng of protein
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Fig.2. Global phosphorylation levels measured in three independently processed samples. (A) rhOPN; (B) protein extracts from cortical bone of 77-year-old Caucasian female donor (77 CF); (C) WT mice; (D) OPN ( / ) C57BL/6 mice. Phosphorylation and dephosphorylation levels are shown as red (upper) and blue (lower) bars, respectively. The baseline corresponds to the native (control) phosphorylation level of bone matrix proteins (change in absorbance on y-axis) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig.3. Natural global phosphorylation levels of bone matrix proteins measured in three age groups of human cadaveric donors: (A) males; (B) females. C, cortical bone; T, trabecular bone; CM, Caucasian male; CF, Caucasian female. Each number preceding “CM” or “CF” represents age in years.
extract for female donors (Fig. 3B). Interestingly, the levels of global phosphorylation of proteins from human trabecular bone were higher than those from cortical bone and were on average 1.24 ± 0.19 and 1.13 ± 0.17 A415/100 ng of protein extract for males and females, respectively. To our knowledge, these are the first observations showing a difference in natural, global levels of phosphorylation between human cortical and trabecular bone. We think that one of the reasons for the dissimilarity in global phosphorylation between cortical and trabecular bone may be higher turnover to remodel trabecular bone as compared with cortical bone. In conclusion, we have developed a strategy that permits modulating and quantitating global phosphorylation of bone matrix proteins that meets different needs of bone tissue engineering and research. Acknowledgments We thank Caren M. Gundberg for bone samples of WT and OPN ( / ) mice and for valuable comments on the manuscript. Research reported in this article was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the
National Institutes of Health under award R01 AR49635. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References [1] K. Engholm-Keller, M.R. Larsen, Technologies and challenges in large-scale phosphoproteomics, Proteomics 13 (2013) 910e931. [2] D.E. Kalume, H. Molina, A. Pandey, Tackling the phosphoproteome: tools and strategies, Curr. Opin. Chem. Biol. 7 (2003) 64e69. [3] M. Mann, A. Pandey, Proteomics to study genes and genomes, Nature 405 (2000) 837e846. [4] E. Salih, H.-Y. Zhou, M.J. Glimcher, Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases, J. Biol. Chem. 271 (1996) 16897e16905. [5] E. Salih, J. Wang, J. Mah, R. Fluckinger, Natural variation in the extent of phosphorylation of bone phosphoproteins as a function of in vivo new bone formation induced by demineralized bone matrix in soft tissue and bony environments, Biochem. J. 364 (2002) 465e474. [6] Z. Lin, S. Cao, X. Chen, W. Wu, J. Li, Thermoresponsive hydrogels from phosphorylated ABA triblock copolymers: A potential scaffold for bone tissue engineering, Biomacromolecules 14 (2013) 2206e2214. [7] A. Iliuk, J.S. Martinez, M.C. Hall, W.A. Tao, Phosphorylation assay based on multifunctionalized soluble nanopolymer, Anal. Chem. 83 (2011) 2767e2774. n, D. Vashishth, Biochemical characterization of [8] G.E. Sroga, L. Karim, W. Colo major bone-matrix proteins using nanoscale-size bone samples and
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[9]
[10] [11] [12]
proteomics methodology, Mol. Cell. Proteomics 10 (2011), http://dx.doi.org/ 10.1074/mcp.M110.006718. G.E. Sroga, P.-C. Wu, D. Vashishth, Link between bone fragility, concentration of IGF1, and glycation: Implications for fracture resistance of bone, PLoS One 10 (1) (2015) e0117046. G.E. Sroga, D. Vashishth, Effects of bone matrix proteins on fracture and fragility in osteoporosis, Curr. Osteoporos. Rep. 10 (2012) 141e150. J. Sodek, B. Ganns, M.D. McKee, Osteopontin, Crit. Rev. Oral Biol. Med. 11 (2000) 279e303. C. Qin, O. Baba, W.T. Butler, Post-translational modifications of SIBLING proteins and their roles in osteogenesis and dentinogenesis, Crit. Rev. Oral Biol. Med. 15 (2004) 126e136.
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[13] B. Christensen, M.S. Nielsen, K.F. Haselmann, T.E. Petersen, E.S. Sørensen, Posttranslationally modified residues of native human osteopontin are located in clusters: Identification of 36 phosphorylation and five O-glycosylation sites and their biological implications, Biochem. J. 390 (2005) 285e292. [14] W.N. Addison, D.L. Masica, J.J. Gray, M.D. McKee, Phosphorylation-dependent inhibition of mineralization by osteopontin ASARM peptides is regulated by PHEX cleavage, J. Bone Min. Res. 25 (2010) 695e705. [15] S. Narisawa, M.C. Yadav, J.L. Mill an, In vivo overexpression of tissuenonspecific alkaline phosphatase increases skeletal mineralization and affects the phosphorylation status of osteopontin, J. Bone Min. Res. 28 (2013) 1587e1598.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A strategy to quantitate global phosphorylation of bone matrix proteins _ Grazyna E. Sroga, Deepak Vashishth* Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2015 Received in revised form 7 January 2016 Accepted 25 January 2016 Available online 3 February 2016
Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured “in bulk” are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials. © 2016 Elsevier Inc. All rights reserved.
Keywords: Global phosphorylation Human bone Mouse bone Extracellular matrix proteins Osteopontin
Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and the landscapes of their phosphorylation in regulation of different cellular functions. Mass spectrometry (MS)1 has become the technique of choice to identify phosphorylated residues within proteins, whereas fragmentation analysis by tandem mass spectrometry (MS/MS) is used to confirm the peptide sequence and to assign the site of modification [1e4]. It is worth noting that the reliable localization of phosphorylation sites in proteins represents one of the most challenging tasks in phosphoproteomic studies. Although powerful, MS tends to be biased toward certain phosphorylated sites, and in general it is not quantitative. Thus, routine analyses of, for example, age-related changes in global phosphorylation levels of proteins
* Corresponding author. E-mail address: [email protected] (D. Vashishth). 1 Abbreviations used: MS, mass spectrometry; MS/MS, tandem mass spectrometry; WT, wild-type; OPN ( / ), osteopontin knockout; CK2, casein kinase II; rhOPN, recombinant human osteopontin; MT plate, microtiter plate; avidineHRP, avidin-linked horseradish peroxidase; PTM, post-translational modification. http://dx.doi.org/10.1016/j.ab.2016.01.017 0003-2697/© 2016 Elsevier Inc. All rights reserved.
using MS equipment are impractical for daily tests performed on patient specimens in a present-day clinical setting. The quantitative measurement of global protein phosphorylation in comparison with the total amount of protein is essential to clinical and pharmacological research and to biomedical applications. From the perspective of bone tissue research, such studies are particularly important for, for example, the understanding of biomineralization regulation occurring in vivo [5] and/or biomineralization performed in vitro for various purposes of bone tissue engineering [6]. Thus, for the needs of bone research and biomedical engineering, we developed a strategy that combines in vitro phosphorylation and dephosphorylation of mineralized bone with an assay for the quantification of global phosphorylation of bone matrix proteins. The phosphorylation levels were measured “in bulk” using a pIMAGO-based system recently developed by Iliuk and coworkers [7]. In the current study, we have, for the first time, quantified global phosphorylation levels of extracellular bone matrix proteins isolated from bone tissues. The samples originated from male and female human cadaveric donors of different ages as well as from wild-type (WT) and osteopontin knockout [OPN ( / )] C57BL/6 mice. To this end, we were able to capture a
86
G.E. Sroga, D. Vashishth / Analytical Biochemistry 499 (2016) 85e89
difference in the global phosphorylation levels between matrix proteins originating from WT and OPN ( / ) mice. We also demonstrated that there is a difference in the levels of global phosphorylation between bone matrix proteins isolated from human cortical and trabecular bone. We expect that the developed strategy has potential to open new avenues for studies on global phosphorylation of bone matrix proteins and their role in bone biomineralization and fragility fractures as well as for other tissues/cells and protein-based materials. Materials and methods Human and mouse bone samples Tibias (posterior area) from a total of 8 human female (4) and male (4) cadaveric donors (young [25.0 ± 2.0 years], middle age [61.0 ± 2.0 years], and elderly [85.0 ± 2.0 years] donors as well as 1 female donor of 77 years) served as the source of cortical and/or trabecular (spatially matched) bone tissue samples. The specimens were obtained from the centralized National Disease Research Interchange (NDRI) biobank and were known to be free of osteoarthritis, diabetes, and other metabolic bone diseases as well as HIV and HVB. Tibias from a total of 3 WT and 3 OPN ( / ) C57BL/6 mice were harvested, cleaned, and cut using a slow-speed diamond blade (Buehler, Lake Bluff, IL, USA). The mice bones served as the source of cortical bone. Both human and mice bone pieces were repeatedly washed in cold distilled water until the washings were free of blood and then were defatted using isopropyl ether. After freeze-drying, the specimens were stored at 80 C until their use. Dephosphorylation and phosphorylation of mineralized bone samples and osteopontin Three types of experiments were performed on bone samples and osteopontin (recombinant human osteopontin, R&D Systems, Minneapolis, MN, USA): phosphorylation, dephosphorylation, and non-enzyme-treated control reactions. Phosphorylation was conducted by incubating the samples overnight at 30 C with casein kinase II (CK2) and the reaction buffer from New England Biolabs (Ipswich, MA, USA). Adenosine triphosphate was added to the buffer as the phosphoryl donor for CK2. The incubation solution also contained 50 mM CaCl2, protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1, Pierce Biotechnology, Rockford, IL, USA) and antibiotics (ampicillin and kanamycin). Dephosphorylation was performed by incubating the samples overnight at 37 C with calf intestinal alkaline phosphatase and the reaction buffer from New England Biolabs. The incubation solution also contained 50 mM CaCl2, protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1) and antibiotics (ampicillin and kanamycin). All control reactions contained only the enzyme incubation buffers supplemented with 50 mM CaCl2, protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1), and antibiotics (ampicillin and kanamycin). Isolation of bone matrix proteins for pIMAGO-based assay Extracellular bone matrix proteins were isolated as described by Sroga and coworkers [8,9] with some modifications to the published procedures. Specifically, in addition to protease inhibitors (100 Protease Inhibitor Cocktail Kit, final concentration 1), a mixture of phosphatase inhibitors (100 Halt Phosphatase
Inhibitor Cocktail, final concentration 1, Pierce Biotechnology) was added to the protein extraction buffer. Quantitation of global phosphorylation levels for extracellular bone matrix proteins and osteopontin using pIMAGO-based assay Quantitative measurement of global phosphorylation levels in protein extracts isolated from each bone sample and all control reactions was performed as described by Iliuk and coworkers [7] using a newly developed pIMAGO phosphoprotein detection system. Briefly, the extracted proteins, recombinant human osteopontin (rhOPN), and phosphorylated casein (additional phosphoprotein control [7]) were bound to the wells of the polystyrene microtiter plates (MT plates). The PBS buffer alone (0.5 and 1) and plasmid DNA alone (0, 10, 20, 40, 80, and 100 ng DNA/ well) were also included in the assay as controls. After a series of blocking (Tris-buffered saline with 5% nonfat dried milk, pH 7.4) and washings (Tris-buffered saline with 0.1% Tween 20, pH 7.4) steps, the pIMAGO reagent (Tymora Analytical, West Lafayette, IN, USA) was added to the MT plate wells to allow the attachment of pIMAGO to the phosphate groups on proteins. The pIMAGO is a water-soluble nanopolymer that is functionalized with titanium(IV) ions that specifically bind to phosphoproteins. The nanopolymer also contains biotin groups that allow the enzyme-linked spectrophotometric detection. In the next step, pIMAGO excess was removed by a minimum of three washings (Tris-buffered saline with 0.1% Tween 20, pH 7.4), and the avidin-linked horseradish peroxidase (avidineHRP) was added to the wells in order to proceed with the colorimetric detection. The absorbance of the solutions in each well was determined at 415 nm using a microtiter plate reader (Infinite 200, Tecan). Results and discussion In vitro dephosphorylation and phosphorylation of mineralized bone samples It was necessary to modify the composition of the buffers supplied with enzymes from New England Biolabs for the needs of the reactions performed on mineralized bone tissues. Thus, to maintain bone's mineral content, 50 mM CaCl2 was used. Preservation of the natural mineral content in bone samples is important if, for example, bone fracture tests were going to follow the phosphorylation/dephosphorylation procedures. To block the activity of proteases, which are naturally present in the extracellular bone matrix, a cocktail of protease inhibitors was added to the reaction buffers. In addition to sterilization of the used solutions (i.e., depending on the compounds, autoclaving or sterile filtration), antibiotics (ampicillin and kanamycin) were also added to prevent the potential growth of bacteria. Isolation of bone matrix proteins for pIMAGO-based assay For the analysis of global protein phosphorylation, protein extracts should reflect as accurately as possible the natural phosphorylation levels of all proteins isolated, for example, at a certain age of a donor or at a particular time point from implanting a graft. Thus, to prevent undesired dephosphorylation of bone matrix proteins after the completion of all the reactions, the cocktail of phosphatase inhibitors was also introduced to the protein extraction buffer in order to block the activity of alkaline phosphatases naturally present in bone matrix.
G.E. Sroga, D. Vashishth / Analytical Biochemistry 499 (2016) 85e89
Specific and highly sensitive measurement of global phosphorylation levels of extracellular bone matrix proteins Before quantifying natural global phosphorylation levels of bone matrix proteins isolated from different human and mice bones, we tested the specificity and sensitivity of the pIMAGO nanopolymer on a smaller set of samples, which included the in vitro phosphorylated and dephosphorylated rhOPN and cortical bone samples originating from the 77-year-old Caucasian female donor. After isolation of extracellular matrix proteins from the bone samples, the respective protein extracts and phosphorylated/dephosphorylated rhOPN samples were bound to the wells of the MT plates. In addition, different concentrations of plasmid DNA alone (0, 10, 20, 40, 80, and 100 ng DNA/well) and PBS buffer alone (0.5 and 1) were bound to the wells of the MT plates (Fig. 1). They served as independent controls of the pIMAGO nanopolymer specificity. Using these additional controls, we demonstrated that the pIMAGO nanopolymer does not bind to the free phosphate groups or DNA. Notably, our protein extraction procedure [8,9] was developed to ensure that there is no detectable DNA or hydroxyapatite in the prepared protein extracts. Still, it was important to show that the aforementioned compounds do not interfere with the determination of the global phosphorylation levels of the extracellular bone matrix proteins when the pIMAGO nanopolymer is used. We were particularly interested in osteopontin because it not only is one of the major phosphoproteins of bone matrix but also is significantly phosphorylated in bone [4,10]. In addition, OPN is extensively altered through other types of post-translational modifications (PTMs) such as glycosylation and proteolytic processing. All PTMs of OPN have significant effects on the structure and biological properties of the protein [11,12]. The nature of OPN phosphorylation is highly tissue and cell specific, reflecting the diverse functions of the protein in different physiological environments. Human OPN has 36 identified phosphorylation sites [13]. The 36 amino acid residues that undergo phosphorylation represent the maximal level of OPN modification because significant variability referring to those 36 phosphorylation sites was observed within and between different cells and tissues [13,14]. Therefore,
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we assumed that due to both the high level of OPN phosphorylation and its high matrix content, even small changes in the global OPN phosphorylation would be less complicated to follow as compared with other less abundant and not as extensively phosphorylated proteins. We demonstrated that under the developed pIMAGO-based assay conditions, the level of global phosphorylation of rhOPN determined before the in vitro phosphorylation was approximately 0.532 ± 0.012 A415/100 ng rhOPN and increased to approximately 1.326 ± 0.004 A415/100 ng rhOPN after the treatment using CK2 (Fig. 2A). Thus, we confirmed that we do have capabilities to alter and measure global phosphorylation levels of a protein. Notably, during our experiments phosphorylation and dephosphorylation of rhOPN was conducted to completion. Therefore, the resulting phosphorylation pattern of rhOPN was most likely different from the in vivo phosphorylation patterns observed for bone matrix OPN [5]. The ability to control and quantify the levels of global phosphorylation of OPN (as well as other phosphoproteins of bone matrix known as SIBLING proteins [12]) is of great interest for the field of bone tissue engineering [4,5] because this phosphoprotein was demonstrated to regulate hydroxyapatite mineralization [14,15]. Next, we showed that the in vitro phosphorylation and dephosphorylation of matrix proteins in human cortical bone of tibia from the 77-year-old Caucasian female indeed changed the natural (0.616 ± 0.004 A415/100 ng of protein extract) levels of phosphorylation (Fig. 2B). Thus, after the in vitro phosphorylation, the global phosphorylation levels of the extracellular bone matrix proteins isolated from three cortical bone pieces of the posterior area were similar and comprised 0.993 ± 0.010 A415/100 ng of protein extract. The in vitro dephosphorylation led to stripping of phosphate groups from the matrix proteins to nearly the same level of 0.228 ± 0.019 A415/100 ng of protein extract. Hence, we established that it is possible to modulate in vitro the levels of bone matrix phosphorylation, an issue that is essential to the field of bone tissue engineering. Moreover, we embarked on the quantitation of global bone matrix phosphorylation in WT and OPN ( / ) knockout mice. We hypothesized that due to the lack of OPN in the OPN ( / ) mice, one could observe a difference in the global phosphorylation level of extracellular matrix proteins isolated from bones of WT and OPN ( / ) mice (Fig. 2C and D). Indeed, we determined that the natural phosphorylation levels were slightly higher in the WT mice bones (0.612 ± 0.010 A415/100 ng of protein extract) when compared with the OPN ( / ) mice bones (0.474 ± 0.015 A415/100 ng of protein extract). Similarly, after the in vitro dephosphorylation, the levels of phosphorylation declined to 0.330 ± 0.010 A415/100 ng of protein extract. Hence, we demonstrated that the pIMAGO-based assay is sensitive enough to detect differences in the global phosphorylation levels between OPN and all other proteins of the extracellular bone matrix. Global phosphorylation levels of extracellular matrix proteins differ between human cortical and trabecular bone
Fig.1. Photograph of an MT plate with the results of the pIMAGO-based assay. The following samples were adsorbed into the wells of the plate. Controls: b-casein (100 ng/well, row A, lanes 1e3), 0.5 PBS buffer (row A, lanes 4e6), 1 PBS (row A, lanes 7e9). Protein extracts from 77-year-old Caucasian female bone samples: dephosphorylated (rows BeD, lanes 1e3), control: native phosphorylation (rows EeG, lanes 1e3), phosphorylated in vitro (rows BeD, lanes 4e6). Recombinant human OPN in triplicate (20, 50, and 100 ng/well): dephosphorylated (rows EeG, lanes 4e6), control: original phosphorylation (rows BeD, lanes 7e9), phosphorylated in vitro (rows EeG, lanes 7e9). DNA in duplicate (0, 10, 20, 40, 80, and 100 ng DNA/well) (rows BeG, lanes 10 and 11).
The developed strategy was ultimately used for the measurement of natural levels of the global protein phosphorylation in the extracellular matrix proteins isolated from the spatially matched cortical and trabecular bone samples isolated from tibias (posterior area) of human female and male cadaveric donors of three age groups (young [25.0 ± 2.0 years], middle age [61.0 ± 2.0 years], and elderly [85.0 ± 2.0 years] donors) (Fig. 3). We established that the natural levels of human cortical bone phosphorylation measured “in bulk” were on average 0.818 ± 0.17 A415/100 ng of protein extract for male donors (Fig. 3A) and 0.663 ± 0.07 ng of protein
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Fig.2. Global phosphorylation levels measured in three independently processed samples. (A) rhOPN; (B) protein extracts from cortical bone of 77-year-old Caucasian female donor (77 CF); (C) WT mice; (D) OPN ( / ) C57BL/6 mice. Phosphorylation and dephosphorylation levels are shown as red (upper) and blue (lower) bars, respectively. The baseline corresponds to the native (control) phosphorylation level of bone matrix proteins (change in absorbance on y-axis) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig.3. Natural global phosphorylation levels of bone matrix proteins measured in three age groups of human cadaveric donors: (A) males; (B) females. C, cortical bone; T, trabecular bone; CM, Caucasian male; CF, Caucasian female. Each number preceding “CM” or “CF” represents age in years.
extract for female donors (Fig. 3B). Interestingly, the levels of global phosphorylation of proteins from human trabecular bone were higher than those from cortical bone and were on average 1.24 ± 0.19 and 1.13 ± 0.17 A415/100 ng of protein extract for males and females, respectively. To our knowledge, these are the first observations showing a difference in natural, global levels of phosphorylation between human cortical and trabecular bone. We think that one of the reasons for the dissimilarity in global phosphorylation between cortical and trabecular bone may be higher turnover to remodel trabecular bone as compared with cortical bone. In conclusion, we have developed a strategy that permits modulating and quantitating global phosphorylation of bone matrix proteins that meets different needs of bone tissue engineering and research. Acknowledgments We thank Caren M. Gundberg for bone samples of WT and OPN ( / ) mice and for valuable comments on the manuscript. Research reported in this article was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the
National Institutes of Health under award R01 AR49635. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References [1] K. Engholm-Keller, M.R. Larsen, Technologies and challenges in large-scale phosphoproteomics, Proteomics 13 (2013) 910e931. [2] D.E. Kalume, H. Molina, A. Pandey, Tackling the phosphoproteome: tools and strategies, Curr. Opin. Chem. Biol. 7 (2003) 64e69. [3] M. Mann, A. Pandey, Proteomics to study genes and genomes, Nature 405 (2000) 837e846. [4] E. Salih, H.-Y. Zhou, M.J. Glimcher, Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases, J. Biol. Chem. 271 (1996) 16897e16905. [5] E. Salih, J. Wang, J. Mah, R. Fluckinger, Natural variation in the extent of phosphorylation of bone phosphoproteins as a function of in vivo new bone formation induced by demineralized bone matrix in soft tissue and bony environments, Biochem. J. 364 (2002) 465e474. [6] Z. Lin, S. Cao, X. Chen, W. Wu, J. Li, Thermoresponsive hydrogels from phosphorylated ABA triblock copolymers: A potential scaffold for bone tissue engineering, Biomacromolecules 14 (2013) 2206e2214. [7] A. Iliuk, J.S. Martinez, M.C. Hall, W.A. Tao, Phosphorylation assay based on multifunctionalized soluble nanopolymer, Anal. Chem. 83 (2011) 2767e2774. n, D. Vashishth, Biochemical characterization of [8] G.E. Sroga, L. Karim, W. Colo major bone-matrix proteins using nanoscale-size bone samples and
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