Liu et al. BMC Musculoskeletal Disorders (2015) 16:281 DOI 10.1186/s12891-015-0730-z RESEARCH ARTICLE Open Access Comparative study of serum proteomes in Legg-Calve-Perthes disease Ruiyu Liu1, Lihong Fan1, Longbin Yin1, Kunzheng Wang1, Wusheng Miao2, Qichun Song1, Xiaoqian Dang1, Hang Gao3 and Chuanyi Bai1* Abstract Background: Legg-Calve-Perthes Disease (LCPD) is an idiopathic osteonecrosis of the developing femoral head complicated by pain and disability of the hip joint. To date, the pathological mechanisms of LCPD are not well-known. This study screened the changes in serum protein expression in patients with LCPD. Methods: Age- and sex-matched serum samples from 10 control subjects and 10 patients with LCPD were compared using the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses, KEGG pathway and functional network analyses were performed. Proteins of interest with large differences in expression, S100-A8, alpha-1-acid glycoprotein 1, haptoglobin and apolipoprotein E, were compared by western blotting. Results: The disease/control ratios showed 26 proteins were significantly differentially expressed (all p < 0.05). Including higher abundances of complement factor H (1.44), complement C4-B (1.45), isocitrate dehydrogenase [NAD] subunit alpha (2.7) alpha-1-acid glycoprotein 1 (1.87), heptoglobin (1.53) and Ig lambda-2 chain C regions (1.46), and lower levels of apolipoprotein E (0.50), apolipoprotein F (0.60), apolipoprotein C-III (0.69), S100-A8 (0.73), S100-A9 (0.75) and prothrombin (0.77) in LCPD than in controls. The alpha-1-acid glycoprotein 1 and haptoglobin increases, and apolipoprotein E and S100-A8 decreases were confirmed by western blot. KEGG pathway analysis revealed these proteins were related to the complement and coagulation cascades, Staphylococcus aureus infection, PPAR signaling, fat digestion and absorption, and vitamin digestion and absorption. Functional network analysis suggested that the proteins were involved in lipid regulation. Conclusions: The complement and coagulation cascades, and abnormal lipid metabolism may be involved in the pathogenesis of LCPD. Keywords: Legg-Calve-Perthes disease, Serum proteome, Isobaric tags for relative and absolute quantification, Lipid metabolism Background developing osteoarthritis in adults [3]. However, the Legg-Calve-Perthes disease (LCPD) is an idiopathic underlying mechanisms and determinants of LCPD are osteonecrosis of the developing femoral head compli- still unclear including the proteins involved. This situation cated by pain and disability of the hip joint. The disease hampers effective treatment and etiological investigation. affects childhood between 6 and 9 years of age. The esti- Coagulation abnormalities [4–6], vascular interruptions mated annual incidence ranges from 0.2 to 19.1 per of the blood supply to the proximal femur [7] and abnor- 100,000 among children under 15 years [1]. It occurs 5 mal immunological reactivity [8] are all linked to the times more often in boys than in girls [2]. The main pathogenesis of LCPD. In addition, it has been suggested long-term problem with this disease is a permanent de- that leptin, a lipid regulator, might play an important role formity of the femoral head, which increases the risk of [9]. Another study showed abnormalities in the vascular structure and function in children with LCPD disease * Correspondence: [email protected] [10]. In experimental animal models, acute bone mor- 1Department of Orthopaedic, the Second Hospital Affilicated to Medical College, Xi’an Jiaotong University, No.157, Xiwu Road, Xi’an, Shaanxi 710004, phogenic protein 2 (BMP2) upregulation [11], increased P. R China vascular endothelial growth factor (VEGF) expression Full list of author information is available at the end of the article © 2015 Liu et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. BMC Musculoskeletal Disorders (2015) 16:281 Page 2 of 9 in the epiphyseal cartilage [12], and increased matrix stage III patients. The healthy children, who had a phys- mineralization [13] in the immature femoral head fol- ical examination before admission to our hospital, were low induction of ischemic osteonecrosis. These findings randomly enrolled to serve as the control group in the imply that multiple pathological reactions may be in- same period by being age- and gender-matched. The re- volved in the development of LCPD. cruitment criteria was as follows: the healthy children Comparative serum proteomic analysis has been used had no disease and bone disease history, with no previ- for years as a potent tool for understanding the pathology ous or current symptoms related to the hip and a nor- of many diseases, especially for those diseases, like LCPD, mal hip radiograph (if available) or no limitation of hip about which we have very limited knowledge. A similar abduction and internal rotation (in children without hip strategy has been applied to osteonecrosis of the femoral radiographs). There were no significant differences in head in adults [14, 15]. Given that these two diseases body weight and height between the two groups. ITRAQ occur in the same location and have similar symptoms the analysis was performed for the first ten patients and ten results of that study should also help us understand the healthy children (clinical characteristics are showed in pathology of LCPD. Isobaric tags for relative and absolute Table 1). The blood sample of another 20 patients and quantitation (iTRAQ), is a quantitative proteomic ap- the age and sex matched healthy children were used to proach with relatively high throughput that allows for sim- performed west-blotting analysis (clinical characteristics ultaneous identification and peptide quantification by are shown in Table 2). measuring the peak intensities of reporter ions with tan- This study was approved by the Ethical Committee of dem mass spectroscopy (MS/MS) that has been developed the Second Affiliated Hospital of Xi’an Jiaotong University, and utilized to identify biomarkers for various disease Xi’an, China. Signed informed-consent documents conditions [16, 17]. This chemical labeling method in- were obtained from all the study participants and volves the stable incorporation of isotopes into an amine their guardians. tagging reagent, which can then be reliably detected by mass spectrometry, thereby permitting comparative quan- Protein Digestion and isobaric tags for relative and absolute titation of various proteins. This method has been sug- quantification (iTRAQ) labeling gested to be suitable for the discovery of differentially Two milliliters of peripheral venous blood was obtained expressed proteins in a wide range of body fluids and from each patient in the outpatient department, and tissues, including serum and plasma [17, 18]. processed to collect serum and then stored at −80 °C for In this study, we performed a comparison of serum further analysis. Fourteen high-abundance proteins in proteomes in child patients with LCPD using the bioin- the serum pools were depleted using a multiple affinity formation match iTRAQ method, to assess the simultan- removal system (Agilent Human 14, Agilent Technolo- eous expression of several serum proteins. This allowed gies, USA) following the manufacturer’s protocol. A 5 us to characterize the associations between differentially KDa ultrafiltration tube (Sartorius) was used for desalin- expressed proteins and the incidence of LCPD. ation and concentration. The protein content of the de- pleted serum was determined by the Bradford assay Methods (Bio-Rad protein assay) according to the manufacturer’s Patients instructions. The study included a total of 20 healthy children and 20 Protein digestion was performed according to the Filter patients with LCPD. All the patients and the gender/age aided sample preparation (FASP) procedure described by matched healthy children were recruited between No- Wisniewski et al. [16]. The resulting peptide mixture was vember 2011 and August 2012 from the second Hospital labeled using the 4-plex iTRAQ reagent (Applied Biosys- affiliated to Xian Jiaotong University (Xi’an, China). The tems). Briefly, 200 μg of peptides from each sample were diagnosis of LCPD was verified based upon the radio- added to 30 μl of STD buffer (4 % sodium dodecyl sulfate graphic appearance and clinical features [19]. The re- (SDS), 100 mM dithiothreitol (DTT), 150 mM Tris–HCl cruited patients were diagnosed with the disease for the pH 8.0). The detergent, DTT and other low-molecular- first time and they had accepted no treatment including weight components were removed using UA buffer (8 M agent, surgery or the other treatments (non-weight bear- ing, bed rest, cast, etc.) before their blood was sampled. Table 1 Clinical characteristics in two groups for iTRAQ analysis Cases of multiple epiphyseal dysplasia, cerebral palsy, Group Patient group Healthy group and developmental hip dysplasia were excluded owing to Sex (female /male) 1/9 1/9