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Quantitative Analysis of Recombinant Human Serum Amyloid A by Isotope Dilution Mass Spectrometry
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摘要: 血清淀粉样蛋白A（SAA）是炎症临床诊断的四大生物标志物之一。目前，国际尚无SAA标准物质，无法实现临床诊断结果的溯源和标准化。选择大肠杆菌重组表达的人血清淀粉样蛋白A作为原料，对其进行定性和定量分析，开展溯源方法研究。采用凝胶电泳法、液相色谱法和质谱法分析SAA纯度与分子量，结果表明SAA原料纯度>98%，分子量11820.09 Da与理论值一致。建立SAA的氨基酸分析-同位素稀释质谱绝对定量方法，在水解时间为8 h时测定水解样品中苯丙氨酸、异亮氨酸、脯氨酸的含量，计算得到SAA溶液的质量浓度为9.32 µg/g（RSD = 0.91%）；建立SAA的特征肽-同位素稀释质谱绝对定量方法，在SAA与胰蛋白酶质量比为1:1，酶解时间为8 h时测定酶解产物中特征肽EANYIGSDK和GPGGVWAAEAISDAR的含量，计算得到SAA溶液的质量浓度为9.41 µg/g（RSD = 0.49%）。两种方法的测定结果经统计学检验一致，无明显精密度差异，定值结果可溯源至SI单位，为SAA标准物质的研制奠定基础。Abstract: Serum amyloid A (SAA) is one of the four inflammation biomarkers in clinical diagnosis. At present, there is no SAA certified reference materials, which cannot achieve traceability and standardization in clinical SAA diagnosis. In this study, the recombinant human serum amyloid A expressed in E. coli was selected as the raw material, and it was qualitatively and quantitatively analysed to study the traceability method. The purity and molecular weight of SAA were analysed by gel electrophoresis, liquid chromatography and mass spectrometry. The results showed that the purity of SAA was >98%, and the molecular weight of 11820.09 Da was consistent with the theoretical value. An absolute quantification method of amino acid analysis-isotope dilution mass spectrometry for SAA was established. The content of phenylalanine, isoleucine and proline in the hydrolysed sample was determined at 8 h of hydrolysis, and the mass concentration of SAA solution was 9.32 µg/g (RSD = 0.91%). An absolute quantification method of signature peptide-isotope dilution mass spectrometry for SAA was established. Within the mass ratio of trypsin and SAA was 1:1 and digestion time was 8 h, the content of peptide EANYIGSDK, GPGGVWAAEAISDAR was detected by IDMS, and the mass concentration of SAA is calculated to 9.41 g/g (RSD = 0.49%). The results of the two methods were consistent by statistical test, and there was no significant difference shown. The quantitative results of this study are traceable to SI units, which lays a foundation for the development of SAA reference materials.
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Key words:
- metrology /
- serum amyloid A /
- reference materials /
- isotope dilution mass spectrometry /
- traceability
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图 5 SAA水解氨基酸的时间变化趋势图
Figure 5. Time-changing trend diagram of SAA hydrolysis to amino acids
图 7 特征肽EK和GR的总离子流色谱图
Figure 7. Total ions chromatogram of signature peptides EK and GR
图 8 不同酶解时间(a)和胰蛋白酶(b)用量对SAA酶解效率的影响
Figure 8. Effect of different (a) reaction time and (b) trypsin usage on SAA digestion reactions
表 1 AAA-IDMS定量分析SAA结果
Table 1. Quantitative detection results of SAA by AAA-IDMS
序号 SAA蛋白浓度（µg/g） Phe Ile Pro 平均值 1 9.53 9.38 9.41 9.44 2 9.52 9.21 9.14 9.29 3 9.47 9.16 9.10 9.24 4 9.52 9.21 9.23 9.32 平均值 9.51 9.24 9.22 9.32 RSD 0.28% 1.04% 1.50% 0.91% 
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表 2 特征肽质谱检测条件
Table 2. Mass spectrometry detection condition of signature peptides
多肽 母离子m/z 子离子m/z 碎裂电压V 碰撞能量eV EK 498.7 406.0 154 12 IS-EK 502.9 414.0 123 11 GR 728.8 340.0 169 30 IS-GR 734.0 256.3 164 26
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表 3 SP-IDMS定量分析SAA结果
Table 3. Quantitative detection results of SAA by SP-IDMS
序号 SAA蛋白浓度（µg/g） EK GR 平均值 1 9.46 9.27 9.37 2 9.34 9.54 9.44 3 9.39 9.33 9.36 4 9.41 9.48 9.45 平均值 9.40 9.41 9.41 RSD 0.53% 1.34% 0.49%
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