Method Development of Efficient Protein Extraction in Bone Tissue for

May 8, 2007 - National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China, Shanghai T...
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Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis Xiaogang Jiang,†,§ Mingliang Ye,† Xinning Jiang,† Guangpeng Liu,‡ Shun Feng,† Lei Cui,*,‡ and Hanfa Zou*,† National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China, Shanghai Tissue Engineering Research and Development Center, Shanghai 200235, China, and School of Medicine, Suzhou University, Suzhou 215007, China Received January 30, 2007

Exploring bone proteome is an important and challenging task for understanding the mechanisms of physiological/pathological process of bone tissue. However, classical methods of protein extraction for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl). A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue. The bone tissue was first demineralized by HCl solution and then extracted using three different lysis buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also collected for proteome analysis. Automated 2D-LC-MS/MS analysis of the collected protein fractions resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific proteins and 15 potential protein biomarkers previously reported were observed in this study. It was demonstrated that the developed extraction method of proteins in bone tissue, which was also the first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone proteome and might be helpful for clarifying the mechanisms of bone diseases. Keywords: protein extraction • bone proteome • shotgun proteomics • tandem mass spectrometry • bone diseases • biomarker discovery

Introduction It is well-known that bone comprises the largest proportion of body’s connective tissue mass. The role of bone is to provide structural support for animal body, to support muscular contraction resulting in motion, to withstand load bearing, and to protect internal organs and also to serve as a mineral reservoir.1,2 At present, bone diseases, such as osteonecrosis, osteofibrosis, and osteoclasis, are very common. Although major progress has been made in the field of bone diseases during the past few years, most severe bone diseases are still unrecoverable from or not adequately treated because of the lack of thorough understanding of pathogenesis in bone diseases. Therefore, it is necessary to explore bone biology * To whom correspondence should be addresed. Prof. Dr. Hanfa Zou, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Tel, +86-411-84379610; fax, +86-411-84379620; e-mail, [email protected]. Prof. Dr. Lei Cui, Shanghai Tissue Engineering Research and Development Center, Shanghai 200235, China. Tel, +86-21-54641663; fax, +86-21-54641587; e-mail, cuileite@ yahoo.com.cn. † Dalian Institute of Chemical Physics, Chinese Academy of Sciences. ‡ Shanghai Tissue Engineering Research and Development Center. § Suzhou University. 10.1021/pr070056t CCC: $37.00

 2007 American Chemical Society

thoroughly for investigating the mechanisms and discovering biomarker of bone diseases. Bone is composed of bone matrix and several distinctly different cell types, such as osteoblast, osteocyte, and osteoclast.3,4 Bone matrix has two components: a mineral part constituted by hydroxylapatite which contributes with 65-70% to the matrix and an organic part which comprises the remaining 25-30% of the total matrix.2 The major constituents of the organic part of bone matrix are collagen and proteoglycans (PGs), which are regarded as structural components. Others are composed of noncollagenous proteins, such as growth factors, cytokines, and bone-specific proteins, which might play a role during the mineralization process, or exhibit a broad array of functions including the control of cell proliferation, cell-matrix interactions, and mediation of hydroxyapatite.5 Because proteins presented in bone tissue are essential for all life processes of bone and are the most important final products of the information pathways, profiling those proteins is vital to understand the bone biology thoroughly. However, proteome research on bone is mainly focused on in vitro systems using bone cells,6-9 such as osteoblasts and osteoclasts, Journal of Proteome Research 2007, 6, 2287-2294

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research articles to determine which proteins are expressed by a cell type under given experimental conditions, but they cannot establish what the actual protein profile of bone is. Recently, the extraction of proteins directly from bone for proteome analysis had been reported.10,11 The extracted proteins were first separated by 2D gel electrophoresis (2DE), the interested spots were then excised, and proteins inside were identified by mass spectrometer (MS). But the analysis of extreme proteins (extremely basic or acidic, extremely small or big, extremely hydrophobic) was still a substantial challenge for 2DE. Shotgun proteomics, which was a high-throughput proteome analysis approach,12-14 could avoid the intrinsic limitations of 2DE. If multidimensional liquid chromatography separations were applied to separate protein digest prior to MS detection in shotgun proteome analysis, thousands of proteins could be easily identified.15-17 Despite an interesting need for large-scale characterization of bone proteome,5 only one paper has been reported to apply shotgun proteomics for proteome analysis of rat bone which resulted in the identification of only 133 proteins.18 Efficient extraction of proteins is one of the most critical issues for proteome analysis.19 Since bone is almost solid, which is physiologically mineralized, the classical protein extraction methods for soft tissues and cells may not be efficient for bone tissue. Therefore, it is necessary to develop efficient methods for protein extraction from bone tissue. In previous reports of bone proteome analysis,10,11,18 the bones were first ground to powder, and the proteins were extracted by incubation of the powder in lysis buffer. However, mechanically breaking bones into powder was laborious, especially for large animal’s bone. More importantly, large amounts of collagens and PGs by the protocol of grinding the undemineralized bone tissue in the lysis buffer would be easily extracted, which impaired detection of low-abundance proteins and strongly affected isoelectric focusing.20 Therefore, an alternative way of demineralizing bone tissue was adopted, and the efficiency for extraction of proteins from demineralized bone tissue was investigated in this study. Because the physiological/pathological characteristic of dog was very similar to that of human and the dog bone was easy to collect, the study on extraction of protein from bone was conducted using bone harvested from dog. A sequential protein extraction protocol to exhaustively extract proteins from bone was developed for comprehensive proteome analysis of bone tissue. Two-dimensional, high-performance liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) was applied to analyze the protein extracts, which resulted in the identification of 2479 proteins.

Materials and Methods Materials. Magic C18AQ (5 µm, 100 Å pore) was purchased from Michrom BioResources (Auburn, CA), and Polysulfoethyl Aspartamide strong cation exchange (SCX, 5 µm, 200 Å pore) was from PolyLC Inc (Columbia, MD). Fused-silica capillaries (50, 75, and 100 µm i.d.) were purchased from Polymicro Technologies (Phoenix, AZ). CHAPS, dithiothreitol (DTT), iodoacetamide, and protease inhibitors cocktail were all purchased from Sino-American Biotechnology Corporation (Beijing). TPCKtreated trypsin was obtained from Sigmaaldrich (St. Louis, MO). Acetonitrile (ACN, HPLC grade) was from Merck (Darmstadt, Germany). All the chemicals were of analytical grade except ACN. All the water used in the experiment was purified with a Mill-Q system (Bedford, MA). Protein Extraction and Proteolysis. The parietal bone fragments (about 10 mm × 10 mm in size, 1 to 2 mm thick) 2288

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Figure 1. Summary of the sequential protein extraction method for bone tissue.

were harvested from skull of adult Beagle canine. The bone fragments were trimmed free of soft tissue and washed to remove contaminants with phosphate buffer saline (PBS), pH 7.4, containing protease inhibitors cocktail overnight at 4 °C. For evaluation of protein extraction efficiency, three different methods were applied to extract proteins from bone. The same lysis buffer containing 6 M guanidine-HCl, 100 mM Tris (pH 7.4), and protease inhibitors cocktail were used for all methods. (1) Protein extraction with demineralized bone: The bone was incubated (0.2 g of bone tissue /mL of solution) at 4 °C overnight in 1.2 M HCl to demineralize bone tissue. The demineralized bone tissue was washed with water and incubated in the lysis buffer for 72 h at 4 °C. The supernatant was collected as Extract A after centrifugation. (2) Protein extraction with undemineralized bone: The undemineralized bone was directly incubated in the lysis buffer for 72 h at 4 °C. The supernatant was collected as Extract B after centrifugation. (3) Protein extraction with grinding: The bone slice (undemineralized) was grinded in the same lysis buffer and then was incubated for 72 h at 4 °C. The supernatant was collected as Extract C after centrifugation. The sequential protein extraction method to exhaustively extract proteins from bone was as follows: 171 mg of the bone slices were first incubated at 4 °C overnight in 1.2 M HCl to demineralize the bone tissue as the procedure for preparation of Extract A. The supernatant was collected as Extract 1 after centrifugation. Then the residue was washed with water and extracted for 72 h at 4 °C in 100 mM Tris, 6 M guanidine-HCl, pH 7.4, containing protease inhibitors cocktail. The supernatant was collected as Extract 2 after centrifugation. The remain was extracted further for 72 h at 4 °C in the extraction solution as above except containing 0.5 M tetrasodium EDTA. The supernatant was collected as Extract 3 after centrifugation. Finally, the rest was incubated in 6 M HCl at 4 °C. The solution was collected as Extract 4 after centrifugation. The schematic diagram of the above extraction method was shown in Figure 1.

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Protein Extraction in Bone Tissue for Proteome Analysis

The following procedures were all the same for all methods. All crude protein extracts were precipitated by acetone precipitation at -20 °C overnight, then the precipitated protein samples were redissolved in a buffer containing 100 mM Tris, 6 M guanidine-HCl, pH 8.1, and protein concentration was determined by the Bradford assay. The protein samples were reduced by DTT and alkylated by iodoacetamide. Then, the solutions were diluted to 1 M guanidine-HCl, and the pH values were adjusted to 8.1. Finally, trypsin was added (trypsin/ protein, 1:50), and the solution incubated at 37 ˚C for 20 h. The tryptic digests were desalted with C18 solid-phase cartridges. LC-MS/MS Analysis. The configuration of 2D separation followed with MS/MS analysis was identical to our previous report.15 The LC-MS/MS system consisted of a quaternary pump, an autosampler, and an LTQ mass spectrometer equipped with a nanospray source. Tryptic digest of 50 µg of bone protein was loaded onto the SCX column. Then, a series of salt elution steps with salt concentrations of 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, and 500 mM NH4Ac were used to elute peptides from the SCX column onto the analytical column. After each salt elution, the two columns were equilibrated for 5 min with buffer A. Then, the peptides retained on the analytical column were separated by ramping a gradient from 5% to 35% buffer B for 120 min, and 35-80% for 15 min. After the column was flushed by 80% of buffer B for 10 min and equilibrated with 5% of buffer B for 15 min, a new cycle started by another peptide fraction displaced with a higher salt concentration from the SCX column. In this experiment, 12 cycles were performed for an entire analysis of 2D-LC-MS/MS. The number of acquired MS/MS spectra was estimated to be about 400 000 for one 2D separation. The spray voltage was set at 1.8 V, and the normalized collision energy was set at 35.0%. The temperature of the ion-transfer capillary was set at 200 °C. All MS and MS/MS spectra were acquired in the data-dependent mode. The mass spectrometer was set so that 1 full MS scan was followed by 10 MS/MS scans on the 10 most intense ions. The dynamic exclusion function was set as follows: repeat count 2, repeat duration 30 s, and exclusion duration 90 s. System control and data collection were done by Xcalibur software version 1.4 (Thermo). Data Analysis. Each digest was analyzed three times by LC-MS/MS, and data analysis was based on the cumulative total proteins identified in three reduplicate analyses. The acquired MS/MS spectra were searched against NCBI Canis familiariz (dog) database (2006 July version) (http://www. ncbi.nlm.nih.gov) using the TurboSEQUEST in the BioWorks 3.2 software suite (Thermo). Reversed sequences were appended to the database for the evaluation of false-positive rate. Cysteine residues were searched as static modification of 57.0215 Da and methionine residues as variable modification of +15.9949 Da. Peptides were searched using fully tryptic cleavage constraints, and up to two missed cleavage sites were allowed for tryptic digestion. The mass tolerances were 2 Da for parent masses and 1 Da for fragment masses. The peptides were considered as positive identification if the Xcorr were higher than 1.9 for singly charged peptides, 2.2 for doubly charged peptides, and 3.75 for triply charged peptides, and ∆Cn cutoff values were g 0.22. False-positive rates (FPR) were calculated by using the following equation, FPR ) 2n(rev)/ (n(rev) + n(forw)), where n(forw) and n(rev) are the number of peptides identified in proteins with forward (normal) and reversed sequence, respectively.16,21 False-positive rate less than

Table 1. Efficiency of Protein Extraction by Utilizing Three Different Protocols (n ) 3)a extract

A

B

C

Efficiency of Protein Extraction 4.27 ( 0.10 0.73 ( 0.04 3.31 ( 0.15 mg protein/g bone tissue) a (A) Protein extraction with demineralized bone; (B) protein extraction with undemineralized bone; (C) protein extraction with undermineralized bone by grinding.

5% was obtained for the peptide identifications by using the above parameters.

Results Method Development for Efficient Protein Extraction. The main composition of bone is the inorganic matrix of calcium salt, that is, hydroxyapatite. As cells in the bone are sandwiched in the inorganic matrix, it is reasonable to believe that the efficiency of protein extraction from bone tissue could be significantly improved if the inorganic matrix is removed before protein extraction. Therefore, the bone tissue was dissected into slices, and they were incubated in 1.2 M HCl to solubilize the inorganic matrix. This process was known as demineralization, and the obtained tissue is referred as demineralized bone.22 The proteins were then extracted by incubation of demineralized bone slices in denaturing buffer of 6 M guanidine-HCl and 100 mM Tris (pH 7.4). For comparison, the undemineralized bone slices were also incubated in the same denaturing buffer for extraction of proteins. The amounts of proteins extracted were determined, and the results are shown in Table 1. As high as 4.27 mg protein/g bone tissue was obtained for the protein extract prepared from demineralized bone (Extract A), while only 0.73 mg protein/g bone tissue was obtained for undemineralized bone (Extract B). The protein yield for undemineralized bone was about 6-fold lower than that of demineralized bone. It was obviously that the removal of mineral composition in bone significantly improved the efficiency for protein extraction. Proteins could also be extracted from bone tissue by grinding the undemineralized bond tissue in the lysis buffer.10,11,18 This protocol was also applied to extract proteins in undemineralized bone in this study. The amount of protein extract was determined to be 3.31 mg protein/g bone tissue (Table 1), which was also lower than that of demineralized bone. It was demonstrated that the demineralization process of bone improved the efficiency for protein extraction significantly. Although the method of protein extraction from demineralized bone was more efficient, demineralization of bone might result in the loss of proteins due to the solublility of some proteins in HCl solution. Thus, the proteins presented in the HCl solution should be also collected. Moreover, high amounts of proteins in bone tissue have not been extracted yet, which would lose much information for bone proteome analysis. And because of the diversity of protein properties, more proteins might be extracted if multiple lysis buffers were used. Therefore, based on these considerations, a sequential protein extraction protocol as shown in Figure 1 was developed to extract proteins from bone for comprehensive proteome analysis. Briefly, 1.2 M HCl was first used to demineralize the bone matrix and extract acid-soluble proteins. Then, the demineralized bone was incubated in the buffer containing 100 mM Tris and 6 M guanidine-HCl (pH 7.4) to extract water-insoluble noncollagenous bone proteins. Third, the residue was extracted by another buffer containing 100 mM Tris, 6 M guanidine-HCl, Journal of Proteome Research • Vol. 6, No. 6, 2007 2289

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Table 2. Quantity of Protein Extracted, Number of Peptides, and Proteins Identified from the Four Protein Extracts Resulted from the Sequential Extraction Protocol protein extracts

quantity of protein (mg)

number of unique peptides

number of unique proteinsa

number of unique proteinsb

Extract 1 Extract 2 Extract 3 Extract 4

0.77 0.64 0.46 1.90

4227 2984 1241 434

1429 1202 617 299

626 355 165 43

a Accepted fully tryptic peptides only. Xcorr at least 1.9, 2.2, and 3.75 for singly, doubly, and triply charged peptide ions, respectively. ∆Cn g 0.22. False-positive rate less than 5%. b In addition of a, identified with at least two peptides per protein.

Figure 2. Venn-diagram showing protein overlap of the four protein extracts as listed in Table 2.

and 0.5 M tetrasodium EDTA (pH 7.4), which was applied to extract water-insoluble noncollagenous bone proteins closely associated with hydroxyapatite crystallites in the mineralized matrix.23 Finally, the remains were further extracted with 6 M HCl. The quantity of proteins presented in the abovementioned fractions was about 0.77, 0.64, 0.46, and 1.90 mg, respectively (Table 2). As expected, there were lots of proteins presented in the HCl solution used to demineralize the bone tissue (Extract 1). Therefore, to prevent loss of proteins, the HCl solution should not be discarded. 2D-LC-MS/MS was applied to analyze the digests of the four protein extracts, and the number of identified peptides and proteins was listed in Table 2. The largest number of proteins was identified from Extract 1 among the four fractions, which further confirmed that the protein presented in the 1.2 M HCl solution should be collected. Although highest amounts of proteins were presented in Extract 4, lowest number of proteins was identified in Extract 4. The main reason of this result might be that high-abundance proteins or protein fragments presented in this fraction suppressed the identification of other proteins. After combining the database searching results of the four extracts, 6202 unique peptides which matched 2479 unique proteins were identified in total (for the complete list, see Supplemental List 1 in Supporting Information). Among the 2479 proteins identified, only 22 (0.9%) were found in all four extracts, while 820 (33.1%), 525 (21.2%), 186 (7.5%), and 65 (2.6%) proteins were uniquely observed in Extracts 1, 2, 3, and 4, respectively (see Figure 2). The low overlap of proteins identified in these fractions indicated that these extraction steps were complementary, and the sequential protein extraction 2290

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protocol for bone outlined in Figure 1 was efficient for generating more comprehensive proteome coverage of bone. To increase the confidence level of protein identification, a minimum of two peptides for the identification of each protein was utilized for the filtering criterion24 besides the criteria described above. It was found that the false-positive rate at protein level decreased to 0.48%. After applying the stringent criteria, 4539 unique peptides derived from 816 unique proteins (for the complete list, see Supplemental List 2 in Supporting Information) were identified. The discussion in the following text was based on 816 unique proteins identified with high confidence. Characterization of Proteins Identified from Bone Tissue. With the presence of high content of PGs and collagen series proteins in bone tissue, one concern is whether these highabundance proteins could suppress the identification of lowabundance proteins. To answer this question, the identified proteins were sorted by the number of identified peptides, that is, the peptide count. Among the top 10 peptide count proteins (top 10 was shown in Supplemental Table 1 in Supporting Information), four proteins including serum albumin, hemoglobin, myoglobin, alpha-2-HS-glycoprotein were serumderived proteins, which were reported to bind to the bone hydroxyapatite crystals in the mineral compartment.25-27 Two proteins including biglycan and creatine kinase were involved in bone growth and differentiations.28,29 Surprisingly, the majority of collagen series proteins and PGs were not observed in the top 10 peptide count proteins, which indicated that the peptides from collagen and PGs did not seriously affect the identification of other proteins in this study. Many bone-specific proteins were identified in the four extracts of dog skull, and most of them were identified in Extract 1 (Supplemental Table 2 in Supporting Information). As noncollagenous proteins only made up 10% of total bone protein content, a majority of these biologically important proteins were relatively low natural abundance proteins; for example, bone morphogentic proteins (BMPs) content in bone tissue was only 1-2 ng/kg wet bone tissue. Among these bone-specific proteins, quite a few proteins embedded in the bone matrix, like osteocalcin, osteonectin, bone sialoprotein, fibronectin, matrix gla-protein (MGP), bone morphogentic proteins (BMPs), growth factors, cytokines, and proteoglycans (like perlecan and biglycan), were identified in this study. Osteocalcin was one of the most abundant noncollagens proteis in bone, comprising up to 20% of the total noncollagen proteins in bone.30 It was produced by mature osteoblasts and primarily deposited in the ECM of skeletal tissue, and its levels reflected the rate of bone formation31 and might be a potential diagnostic marker for bone diseases such as oteoporosis.32 Matrix Gla-protein (MGP), γ-carboxyglutamic acid (GLA)-rich, vitamin K-dependent, and apatite-binding protein were the regulator of bone and cartilage mineralization during development.33 Moreover, BMPs possessed the greatest in vivo bone stimulatory capacity and were recognized as the only growth factors to simulate mesenchymal stem cells to differentiate along osteoblastic and chondrogenic lineages,34 which promoted the formation of bone and the skeleton and mended broken bones.35,36 Other noncollagenous glycoproteins in bone matrix, such as osteonectin, fibronectin, osteopontin, and bone sialoprotein (BSP), which comprised 15% of the total noncollagens proteins in bone,30 were also identified, which were produced at different stages of osteoblast maturation. Those identified proteins exhibited a broad spectrum of func-

Protein Extraction in Bone Tissue for Proteome Analysis

tions including the control of cell proliferation, cell-matrix interactions, and mediation of hydroxyapatite deposition. Furthermore, many proteins (cathepsin, matrix metalloproteinases (MMPs), or plasminogen, etc.) associated with bone matrix degradation were observed. The papain family of cysteine proteases, like cathepsin A, D, G, K, was also identified. It was reported recently that cathepsin K was the major protease responsible for bone resorption above all cathepsins mentioned.37 Two MMPs of MMP-2 and MMP-19 identified could destroy all the proteins of the ECM.38 MMP-2, also previously known as the Type IV collagenase,39 had substrate specificity to denature collagens, Type V, VII, X, XII collagens, vitronectin, aggrecan, galectin-3, and elastin, and most of them were also identified. Various substrates of MMP-19 have been identified in vitro40, including type IV collagen, gelatin, laminin1, nidogen 1, fibronectin, and tenascin-C, which were also mostly detected. The identification of these bone-specific proteins demonstrated the effective of the sequential protein extraction protocol developed in this study. To furthur characterize the proteins identified from bone, GoMiner program41 was used to classify the identified proteins, which provided a general view of protein location and function. 1. Subcellular Location. Totally, 301 identified proteins (36.9%) were annotated in Subcellular Location. As shown in Figure 3A, the identified proteins were found to arise from every cell compartment. Moreover, the intracellular locations of the identified proteins were shown in Figure 3B. As illustrated, the majority of the identified proteins (62.7%) originated from the cytoplasm. The next two significant locations were cytoskeleton (29.9%) and mitochondrion (14.9%). In addition, 47 membrane proteins were also identified, as shown in Supplemental Table 3 in Supporting Information. The membrane proteins mediated many vital cellular processes and transferred metabolites and transmitted signals between cells and their environment, between organelles within the cell, and between organ systems.42,43 Despite their importance to cell function and the fact that roughly 20-30% of all open reading frames were predicted to encode for membrane proteins,44 this class of proteins still presented a challenge for proteome analyses because of the difficulties in extraction, solubilization, and separation.43 The results of cellular distribution indicated comprehensive protein information of dog bone was obtained by shotgun proteome analysis. 2. Protein Role Assignment. Totally, 379 identified proteins (46.4%) were annotated in Protein Functions. A graphical representation of identified proteins based on their known physiological functions was presented in Figure 4. Although the largest number of proteins was assigned to binding function, in many cases, it was believed that the function was auxiliary to more important roles of that protein which could be related to other functions. Second, 152 of the identified proteins were engaged in catalytic activity. And additional 28 proteins were classified as their regulators. This indicated that 180 unique proteins identified from the bone proteome related to enzymatic activity. Of all identified enzymes, 61 belonged to the class of hydrolases, which included 18 peptidases. These digestive enzymes needed to be strongly regulated to prevent unwanted proteolysis, and so 24 protease inhibitors were identified in our study, of which 19 were the endopeptidase inhibitors (Supplemental Table 4 in Supporting Information). In addition, 3 kinase inhibitors (Alpha-2-HS-glycoprotein, 143-3 protein gamma, 14-3-3 protein theta), 2 phospholipase inhibitors (Annexin A1, Annexin A5), and 1 phosphatase

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Figure 3. Cellular distribution of the proteins identified from dog skull. (A) The cellular compartment distribution; (B) the intracellular component distribution.

Figure 4. Functional distribution of the proteins identified from dog skull.

inhibitor (Pleiotrophin) were also identified, which were key elements of most signal transduction pathways that regulated cell physiology and pathology.45 Totally, 76 structural proteins were identified in our study, forming the third largest functional group. This group conJournal of Proteome Research • Vol. 6, No. 6, 2007 2291

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Figure 5. Biological process distribution of the proteins identified from dog skull.

tained 22 structural constituents of cytoskeleton, 9 ECM structural constituents, and 2 structural constituents of bone. Moreover, some low-abundance proteins associated with important functions such as protein biosynthesis were also identified in the proteome analysis of dog bone, which included 5 transcription regulator proteins (splicing factor 1, activated RNA polymerase II transcriptional coactivator p15, GLI-Kruppel family member GLI3 isoform 1, YY1 transcription factor isoform 1, zinc finger protein 91) and 3 translational regulator molecules (Elongation factor 2, Tu translation elongation factor, eukaryotic translation initiation factor 5A isoform 2). 3. Biological Process. Totally, 336 identified proteins (41.2%) were annotated in Biological Process. The classification of the identified proteins involved in different biological process categories is shown in Figure 5. The largest category was metabolism which was composed of 198 proteins. This broad category contained many of the above-mentioned enzymes, notably, proteases, as well as enzymes involved in basic cellular processes such as protein catabolism (17 proteins, as shown in Supplemental Table 5 in Supporting Information). A large group of 76 proteins was assigned a role in organismal physiological process. Among those proteins, 8 bone remodeling proteins (secreted phosphoprotein 24, osteocalcin, bone sialoprotein-2, osteonectin, fetuin-A, matrix gla-protein, secreted phosphoprotein 1, sclerostin) were identified. In the third largest group, 59 proteins associated with response to stimulus activity were observed. This group could be categorized into four types: response to biotic stimulus (39 proteins), response to endogenous stimulus (3 proteins), response to external stimulus, and response to stress (42 proteins). Besides proteins involved in the above-mentioned biological processes, there were 55, 55, and 43 proteins involved in the biological processes of localization, transportation, and regulation, respectively.

Discussion Efficient extraction of proteins from bone tissue was critical for bone proteome research. In this study, protein extraction from undemineralized and demineralized bone was compared. It was found that the extraction efficiency was improved significantly after the bone was demineralized by HCl solution. However, demineralization of bone tissue by HCl solution also resulted in the dissolution of acid-soluble proteins in demineralized solution. To prevent the sample loss, the proteins in the HCl solution should also be collected for proteome 2292

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analysis. One concern was the degradation of protein in strong acid. This was a serious problem for the classic protein analysis approach where separation and detection took place at the protein level. However, this was not a problem for the shotgun proteomics approach where the separation and detection took place at the peptide level. Although the proteins were degraded into several fragments, the identifications of proteins could still be achieved by tryptic peptides cleaved from these protein fragments in shotgun proteomics. This was confirmed by the fact that as many as 1429 proteins were successfully identified from the 1.2 M HCl solution for the demineralization of bone. The previously reported protein extract method by grinding the bone tissue was laborious and maximized the extraction of collagens and PGs which strongly affected isoelectric focusing and detection of low-abundance proteins.20 The extraction of proteins by demineralization of bone tissue is more simple and efficient as only a few incubation steps were conducted and minimized extraction of collagens and PGs which was in favor of bone proteome profiling. Acid-soluble proteins were extracted from bone tissue during demineralization, and other proteins could be extracted by different lysis buffers. For large-scale proteome analysis, it was preferable to digest these protein fractions and analyze the resulting digests separately. Here, a sequential protein extraction protocol was developed to extract proteins from bone tissue, and proteins in each fraction were analyzed by 2D-LCMS/MS-based proteomics. The low overlap of the proteins identified from these fractions demonstrated the complementarity of these lysis buffers. With the combination of all the database search results, the identification of 6202 unique peptides which matched 2479 unique proteins was obtained with a false-positive rate of 5%. This was the largest data set obtained for bone proteome. However, membrane proteins in bone were not efficiently extracted because of their poor solubility in the lysis buffers used in this study. To further increase the proteome coverage, the composition of lysis buffers should be optimized in the future. The interference of high-abundance proteins in bone such as collagen and PGs on proteome analysis was minimized using this approach. The classification of identified proteins by GoMiner revealed many extracellular and intracellular proteins, and mapped to a broad coverage of molecular function. In addition, an important potential application for proteome analysis was biomarker discovery. Fifteen potential biomarkers of bone diseases detected in the serum, synovial fluid, and so forth46-51 were also found in our study as shown in Supplemental Table 6 in Supporting Information. This indicated that these proteins also presented in bone tissue and could be efficiently extracted by this protein extraction approach.

Conclusion A sequential protein extraction method was developed to large-scale proteome analysis of bone tissue. The protein extraction efficiency was improved significantly by demineralization of bone tissue. The in-depth proteome analysis of bone revealed over 800 proteins based on two peptides minimum. This method was developed for extraction of proteins from dog bone tissue; it should also be easily applied to any animal bone including humans. The technique developed in this study enabled in-depth analysis of protein expression in bone tissue, which was very important to gain insight into the mechanisms of physiological/pathological process of bone tissue.

Protein Extraction in Bone Tissue for Proteome Analysis

Acknowledgment. Financial supports from the National Natural Sciences Foundation of China (No. 20327002), the China State Key Basic Research Program Grant (2005CB522701), the China High Technology Research Program Grant (2006AA02A309), the Knowledge Innovation program of CAS KJCX2.YW.HO9, and the Knowledge Innovation program of DICP to H.Z. and National Natural Sciences Foundation of China (No. 20605022) to M.Y. are gratefully acknowledged. Note Added after Print Publication. Figure 2 was incorrectly produced and, thus, the numbers obtained from it were also incorrect in the version published on the Web May 8, 2007 (ASAP) and published in the June 2007 issue (Vol. 6, No. 6, pp 2287-2294). The figure was replaced and the corresponding numbers in the text were changed; the corrected version was published on the Web September 25, 2007, and an Addition and Correction appears in the November 2007 issue (Vol. 6, No. 11).

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