Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.
Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.
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DOI：
10.1016/j.ymeth.2016.01.013
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发表时间：
2016-07-15
期刊：
影响因子：
--
通讯作者：
Ogorzalek Loo RR
中科院分区：
文献类型：
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作者：
Lohnes K;Quebbemann NR;Liu K;Kobzeff F;Loo JA;Ogorzalek Loo RR
虚拟二维凝胶电泳法/质谱仪(虚拟二维凝胶电泳法/质谱仪)技术结合了二维凝胶电泳法的卓越、高分辨率能力和质谱仪(MS)的灵敏度和高质量准确度。等电聚焦(IEF)凝胶电泳分离的完整蛋白质通过基质辅助激光解吸/电离(MALDI)MS从固定化pH梯度(IPG)聚丙烯酰胺凝胶(经典2D-PAGE的第一维)中成像，并且需要从镶嵌在2D-PAGE凝胶中或IPG条带中的亚皮摩尔水平的蛋白质中获得准确的完整质量，以解释当胰蛋白肽图谱不能解决问题时，2D凝胶上被鉴定为蛋白质A的一个点与另一个被鉴定为同一蛋白质点的蛋白质有何不同。然而，这项任务极具挑战性，实际上很少有人尝试。虚拟2D Gel/MS提供了访问这些完整质量块的途径。对我们的基质沉积程序的修改提高了制备IPG凝胶的可靠性；描述了新的程序。描述了这种高通量的MALDI MS成像(MSI)方法的开发，该方法与集成的“自上而下”的MS一起从复杂的生物样品中阐明了蛋白质异构体，并证明了现在可以在大约5分钟内对4厘米长的IPG凝胶片段进行成像。凝胶范围的化学和酶方法以及MALDIMS/MS的进一步询问提供了鉴定、序列相关信息和翻译后/转录修饰信息。基于MSI的虚拟2D Gel/MS平台可能会将“自上而下”和“自下而上”蛋白质组学的优点联系起来。
The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein ‘A’ on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging and is, in fact, rarely attempted. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput with integrated ‘top-down’ MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5 minutes. Gel-wide chemical and enzymatic methods with further interrogation by MALDIMS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of ‘top-down’ and ‘bottom-up’ proteomics.