Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
Anal Biochem. 2011 Apr 1;411(1):80-7. doi: 10.1016/j.ab.2010.12.010. Epub 2010 Dec 10.
Many basic proteins (pI>7) and putative disease biomarkers are not identified using conventional proteomic methods. This study applied a new method to improve the identification of such proteins. Prefractionated basic proteins were compared with total tissue lysates from human ductal carcinoma in situ tissue loaded on basic immobilized pH gradient strips prior to two-dimensional gel electrophoresis (2-DE). Extraction of alkaline proteins was achieved in less than 20 min using a chromatofocusing resin and two buffers in a microcentrifuge tube. Prefractionation showed improved resolution and visualization of low-abundance proteins on 2-DE gels, allowing proteins to be excised, accumulated, trypsin-digested, and identified by liquid chromatography-tandem mass spectrometry. Proteins identified in the prefractionated samples had a higher number of peptides and three times the number of unique basic proteins when compared with total lysates. Low-molecular-weight (LMW, <26kDa) unique alkaline proteins comprise 75% of those identified in prefractionated samples compared with 25% identified in total lysates, representing a 9-fold increase of LMW proteins due to prefractionation. Prefractionation ultimately increases loading capacity of samples onto the 2-DE gel and leads to better resolution, visualization, and identification of proteins with pI values greater than 7.
许多基础蛋白（pI>7）和潜在的疾病生物标志物都无法使用常规蛋白质组学方法进行鉴定。本研究应用了一种新方法来改善此类蛋白质的鉴定。在二维凝胶电泳（2-DE）之前，将预分级的碱性蛋白与原位导管癌组织的总组织裂解物加载到碱性固定 pH 梯度条上进行比较。使用层析聚焦树脂和两个缓冲液在微量离心管中，在不到 20 分钟内即可提取碱性蛋白。预分级可改善低丰度蛋白在 2-DE 凝胶上的分辨率和可视化，从而可以对蛋白进行切胶、积累、胰酶消化，并通过液相色谱-串联质谱进行鉴定。与总裂解物相比，在预分级样品中鉴定出的蛋白具有更多的肽和三倍数量的独特碱性蛋白。与总裂解物中鉴定出的 25%相比，在预分级样品中鉴定出的低分子量（LMW，<26kDa）独特碱性蛋白占 75%，由于预分级，LMW 蛋白增加了 9 倍。预分级最终增加了样品在 2-DE 凝胶上的上样容量，并提高了 pI 值大于 7 的蛋白的分辨率、可视化和鉴定。
