【摘要】 目的：探讨用表面增强基质辅助激光解析离子化飞行时间质谱（SELDITOFMS )筛选视网膜母细胞瘤（retinoblastoma，Rb）血清标志蛋白，为Rb筛查、早期诊断及鉴别诊断寻找新的方法。方法：采用IMAC30和CM10两种蛋白芯片对18例Rb患者和17名年龄匹配的正常小儿血清进行了蛋白质谱检测。统计分析采用Ciphergenproteinchip3.0.2软件，两组间蛋白峰强度的比较采用t检验，率的比较采用Fisher精确概率法。结果：IMAC30蛋白芯片结果显示：与正常幼儿相比，视网膜母细胞瘤患者血清中有26个差异表达的蛋白峰，其中高表达有21个，质/荷比（ m/z）分别为：7746，7014，11713，3049，7084，7299，5888，2544，12575，5489，9658，9575，9929，10161，8955，1886，10617，6209，2411，7374和6614；低表达有5个，分别为：8382，7923，7972，8590和66576，统计学检验P<0.01。其中以m/z7014 峰进行两组统计学分析时，敏感性和特异性分别为94.4%和82.4%。与正常小儿相比，经CM10蛋白芯片分析显示：视网膜母细胞瘤患者血清中有4个差异表达的蛋白峰，其中高表达有3个，质/荷比（ m/z）分别为5888，6097和7798；低表达蛋白峰为m/z8590 。统计学检验P<0.01。当选择m/z7798 峰进行统计学分析时，敏感性和特异性分别是83.3%和70.6%。结论：视网膜母细胞瘤患者血清中存在较多标志蛋白，SELDITOFMS蛋白质芯片技术有望成为Rb筛查、早期诊断及鉴别诊断的新的有效工具。
【关键词】 视网膜母细胞瘤；SELDI蛋白质芯片技术；差异蛋白
Correspondence to: DaCheng He. Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Universities Confederated Institute of Proteomics, Beijing 100875, [email protected]; Ping Zhang. Zhongshan Ophthalmic Center, Sun Yatsen University, Guangzhou 510060, Guangdong Province, China. [email protected]
INTRODUCTION
At present, the diagnosis and differential diagnosis of retinoblastoma（Rb） are mainly based on the history of the diseases, clinical manifestation, computer tomography(CT) and type B ultransonography and other imaging methods. Some scholars have studied the diagnostic value of the serum neurone specific enolase(NSE) in Rb and the results showed that the sensitivity and specificity of NSE were limited[1,2]. So it is an essential and urgent task to find a better method that has higher sensitivity and specificity in the early diagnosis and screening of Rb.
Surfaceenhanced laser desorption/ionization timeofflight mass spectrometry(SELDITOFMS) is a new, convenient and rapid method for the relative expression levels of proteins over a wide range of molecular weights in biological samples under different conditions. Compared with other proteomic technology, it has many advantages, such as rapid way, high throughput, high sensitivity and specificity, low sample requirement. Furthermore, It can detect multiple changes of different proteins simultaneously, in particular lowerabundance proteins and some lower molecular weight proteins. As a result, it can be applied directly to detect complicate biological specimen like serum[35]. Our purpose is to look for protein markers in sera of Rb patients and to provide new markers for the screening, diagnosis and differential diagnosis of Rb. In addition, it can provide significant information for diagnostic accuracy of Rb and further determination of the candidate proteins in research.
MATERIALS AND METHODS
Serum Samples Thirtyfive serum samples were taken from the Zhongshan Ophthalmic centre of Sun Yatsen University. Among them, seventeen were taken from healthy children and eighteen were taken from Rb patients confirmed by histologic examination. The healthy control group ( n=17, age range 8 months to 8 years, mean 2.9 years) and Rb group( n=18) were agematched. The Rb group was subsequently divided into the intraocular RB group ( n=14) and the extraocular RB group ( n=4).The parents of all subjects had given written informed consent to the study protocol, which was approved by the Ethics Committee of Sun Yatsen University. All serum samples were collected in the morning before breakfast. The sera were left at 4℃ for 1 hour, centrifuged at 4000rpm for 20 minutes and then stored at 80℃.
Reagents and Instruments Carbamide, acetonitrile, trifluoroacetic acid, sinapic acid (SPA), TrisHCl and HEPES were purchased from Sigma Corporation (USA). The ProteinChip Biosystems (Ciphergen PBS II plus SELDITOFMS), CM10 chip and IMAC30 chip were purchased from Ciphergen Biosystems Corporation (USA).
ProteinChip Array Analysis Immobilized metal affinity capture array 30 (IMAC30) were used and put into a bioprocessor. According to the reference[6], the chips were coated with 50μL of 100mmol/L CuSO4 on each array and agitated for 5 minutes at room temperature, and then CuSO4 was discarded. The chips were rinsed three times with deionized H2O. 150μL of 100mmol/L sodium acetate buffer (pH 4.0) was added to each microarray, which was shaken for 5 minutes to remove the unbound Cu2+. 5μL of serum was mixed with 10μL of 8mol/L urea and was vibrated for 30 minutes at 4℃. 10μL of the serum/urea mixture was applied to each array with 90μL of 100mmol/L sodium acetate buffer, incubated and shaken for 30 minutes. After the serum/urea mixture was discarded, the chips were washed twice, 5 minutes in each wash cycle, with 150μL of 100mmol/L sodium acetate buffer as described above. The chips were removed from the bioprocessor, washed twice with deionized H2O, airdried, and stored at room temperature until subjected to SELDITOF analysis.
At the same time, we employed the CM10 Chip for detection. The chip was coated with 5μL serum and 10μL of 9mol/L urea. Above 5μL mixture was added to CM10 with 60μL of 100mmol/L sodium acetate buffer (pH 4.0) and 10μL of 10mmol/L HCl, placed in room temperature for 10 minutes. After washed twice, the chip was installed to Bioprocessor. Next, CM10 chip was washed with 200μL buffer and shaken for 5 minutes, repeated again. 100μL serum sample was added to each spot on the chip , agitated and incubated for 1 hour at room temperature, discarded, then washed and agitated three times, 5 minutes every time. After each spot was washed with 200μL of 1mmol/L HEPES(pH 7.0) and airdried, 0.5L SPA was applied to each spot, repeated twice, airdried again until subjected to SELDITOF analysis.
Before SELDITOF MS analysis, the instrument was calibrated with standard polypeptide and protein marker of lower molecular mass to ensure deviation at 0.05%. Chips were analyzed by the PBSⅡ plus mass spectrometer reader. Data were obtained by averaging the results from a total of 124 laser shots with an intensity of 210, a detector sensitivity of 9, a high mass of 150kDa and an optimized range of 3k30kDa. Peaks were identified after mass calibration, and background subtraction and normalization were realized through the clustering and alignment function of ProteinChip Biomarker Wizard software 3.1 (Ciphergen Biosystems, Fremont, CA, USA).
Bioinformatics and Biostatistics Comparison between groups was performed by analysis of ttest with the ProteinChip Biomarker Wizard software (Ciphergen Biosystems, Fremont, CA, USA). Values of P<0.01 were considered statistically significant. Fishers exact test was used to compare the predominance of differential protein peaks appeared in patients and normal children and the testing standard was set at α=0.05.
RESULTS
Two different chips, IMAC30 and CM10, were performed to analyze serum protein mass spectrogram of 18 Rb patients and 17 normal children. The results showed that there were different peaks between them.
In Figure1A, IMAC30 protein chip showed the protein masses between 7250m/z and 8800m/z. The protein peak of 7746m/z was highly expressed in Rb group and the protein peak of 8382m/z was lowly expressed in healthy individuals. In Figure1B, CM10 protein chip showed the protein masses between 5000m/z and 10000m/z. The protein peak of 5888m/z was highly expressed in Rb group and the protein peak of 8590m/z was lowly expressed in healthy individuals. The X axis represents the molecular mass; the Y axis represents relative peak intensity. RB represents retinoblastoma patients and N represents normal children.
We analyzed the distribution of the differential protein between Rb patients and normal children. From the distribution we can know that there was manifest regularity. For instance, in RB group, the peak intensity at 7014 m/z and 7084m/z by IMAC30 protein chip increased obviously, while the peak intensity at 7054 m/z had no significant difference between RB group and control group(Figure 2A). Otherwise, the peak intensity at 6097m/z by CM10 protein chip was upregulated noticeably in Rb group (Figure 2B).
As Figure 2 showed, the content of proteins of
7014,7084 and 6097m/z increased obviously in Rb patients, but there was no statistically significant difference about content of the protein of 7054m/z between Rb patients and normal children. Figure 2A showed the result of IMAC30 chip and Figure 2B showed the result of CM10 chip. RB represents retinoblastoma patients and N represents normal children. The X axis represents the molecular mass; the Y axis represents logarithm of relative peak intensity.
Our research results showed that 26 differential proteins were detected in the sera of RB patients by contrast with healthy children by using IMAC30 protein chip. Of the 26 proteins, 21 differential proteins were upregulated: 7746, 7014, 11713, 3049, 7084, 7299, 5888, 2544, 12575, 5489, 9658, 9575, 9929, 10161, 8955, 1886, 10617, 6209, 2411, 7374, 6614m/z and 5 differential proteins were downregulated: 8382, 7923, 7972, 8590, 66576m/z. Through statistical analysis, all P values were less than 0.01(Table 1). According to the result of CM10, four differential proteins in RB patients were detected. Among them, three differential proteins increased and one protein decreased in the sera of RB patients. Statistically, all P values were less than 0.01(Table 2). In addition, we respectively used the differential proteins of 7014m/z and 7798m/z to carry out a statistical test, and results showed that the former had a specificity of 82.4% and a sensitivity of 94.4%, and the latter had a specificity of 70.6% and a sensitivity of 83.3%.
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