Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan.
J Pharm Biomed Anal. 2013 Sep;83:293-8. doi: 10.1016/j.jpba.2013.05.031. Epub 2013 May 27.
A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes.
需要结合多种方法来实现完整蛋白质的分离，然后进行结构分析以检查其不稳定或外部结构。本研究的目的是开发一种完整蛋白质结构分析的方法。通过非变性二维电泳（2-DE）产生的膜固定抗转铁蛋白抗体捕获人血浆中的转铁蛋白，并将其转移到聚偏二氟乙烯（PVDF）膜上，并用丽春红 S 染色。抗原转铁蛋白通过用三氟乙酸（TFA）或天冬氨酸冲洗膜洗脱。此外，还建立了一种方法，通过该方法，在基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）靶板上用酶消化可对纯化的人转铁蛋白进行消化。因此，用人血浆中的数十微升免疫亲和膜纯化人转铁蛋白抗原后，在用 0.1%TFA 或胃蛋白酶、内肽酶 Lys-C 或 0.001%SDS 中的 Lys-C/胰蛋白酶消化后，在板上获得转铁蛋白多肽片段。结果表明，免疫亲和膜分离和 MALDI-TOF MS 板上酶消化的联合方法可用于抗原的纯化和微分析。这种方法特别适用于从有限的样品量分析抗原的一级结构和不太稳定和高度可及的区域。
