Department of Endocrinology, Hospital Universitario Ramon y Cajal and Universidad de Alcala and Instituto Ramon y Cajal de Investigacion Sanitaria IRYCIS, Spain.
Proteomics Clin Appl. 2010 Sep;4(8-9):739-44. doi: 10.1002/prca.201000007.
To analyze the impact of storage temperature on the 2D-DIGE profile of human plasma.
2D-DIGE and MS were used to identify the differences in the proteomic profiles of aliquots of eight human plasma samples stored at either - 30 or - 80°C for 18 months.
After the depletion of albumin and IgG, 2D-DIGE identified four spots significantly and reproducibly increased, and five spots that were decreased, in samples stored at - 30°C compared with those stored at - 80°C. These nine spots were manually excised, digested ingel and analyzed by MALDI-TOF/TOF. MASCOT database search using the PMF spectra allowed the identification of the proteins present in eight of the nine spots. All the spots corresponded to the complement C3 precursor protein.
Our present results indicate that plasma collections stored at -30°C, and not only those stored at - 80°C, may be used for 2D-DIGE analyses of albumin-and IgG-depleted human plasma without loosing the essential information about highly abundant proteins. This finding expands the applicability of 2D-DIGE to the study of human disease by permitting the analysis of human plasma samples stored at temperatures between - 30 and - 80°C.
分析储存温度对人血浆 2D-DIGE 图谱的影响。
使用 2D-DIGE 和 MS 技术，鉴定在-30°C 或-80°C 下储存 18 个月的 8 个人血浆样本等分试样的蛋白质组图谱差异。
在耗尽白蛋白和 IgG 后，与在-80°C 储存的样本相比，在-30°C 储存的样本中，2D-DIGE 鉴定出四个明显且可重复增加的斑点，和五个减少的斑点。这些九个斑点被手动切除，胶内消化后，通过 MALDI-TOF/TOF 进行分析。使用 PMF 光谱对 MASCOT 数据库搜索，允许鉴定出九个斑点中的八种蛋白质。所有斑点均对应于补体 C3 前体蛋白。
本研究结果表明，在-30°C 储存的血浆收集物，而不仅是在-80°C 储存的收集物，可用于白蛋白和 IgG 耗尽的人血浆的 2D-DIGE 分析，而不会丢失关于高丰度蛋白质的重要信息。这一发现扩大了 2D-DIGE 在人类疾病研究中的适用性，允许在-30 至-80°C 之间温度下储存的人类血浆样本进行分析。
