Antigen digestion on the target plate of MALDI-TOF MS after isolation using an immunoaffinity membrane.
Antigen digestion on the target plate of MALDI-TOF MS after isolation using an immunoaffinity membrane.
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DOI：
10.1016/j.jpba.2013.05.031
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发表时间：
2013-09
影响因子：
3.4
通讯作者：
Y. Shimazaki;Yuri Nishimura;Masaki Saito
中科院分区：
文献类型：
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作者：
Y. Shimazaki;Yuri Nishimura;Masaki Saito
需要结合多种方法来实现完整蛋白质的分离，并随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纯化后完整蛋白质的结构分析方法。人血浆中的转铁蛋白被通过非变性二维电泳 (2-DE) 产生的膜固定化抗转铁蛋白抗体捕获，转移到聚偏二氟乙烯 (PVDF) 膜上，并用丽春红 S 染色。用三氟乙酸 (TFA) 或天冬氨酸冲洗膜，洗脱抗原转铁蛋白。此外，还建立了在基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）靶板上酶消化纯化的人转铁蛋白的方法。因此，使用免疫亲和膜从数十微升人血浆中纯化人转铁蛋白抗原后，在0.1% TFA或内切蛋白酶Lys-C或Lys-C/胰蛋白酶和0.001%十二烷基硫酸钠(SDS)存在下用胃蛋白酶消化后，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板酶消化相结合的方法可应用于抗原的纯化和微量分析。这种方法特别适用于从有限的样品量中分析抗原的一级结构和不太稳定且容易接近的区域。
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.