Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS
Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS
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DOI：
10.1021/pr015519o
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发表时间：
2002-03-01
影响因子：
4.4
通讯作者：
Karger, BL
中科院分区：
文献类型：
--
作者：
Rejtar, T;Hu, P;Karger, BL
高分辨率毛细管电泳联用MALDI-TOF和TOF/TOF MS，通过离线真空沉积到标准的不锈钢MALDI靶上。这种离线方法允许分离与MS分析分离，从而允许在时间上分别进行独立优化。使用BSA胰酶消化作为模型样品，沉积的条纹，大约100微米宽，首先在MS模式下进行分析，只消耗样品的一小部分。在数据分析后，使用MALDI-TOF/TOF MS在MS/MS模式下对含有未知多肽的沉积痕迹以及选择用于序列确认的几个物种进行了重新分析。此外，结果表明，真空沉积痕迹(5%RSD)的每一次重现性比标准干液滴方法的结果低一个数量级。此外，对于浓度从1到1000 nm的多肽样品，发现信号强度(相对于内标)在3个数量级上的线性相关性。本文展示了利用真空沉积将高分辨率分离与MALDI-MS和MALDI-MS/MS离线耦合用于分析蛋白质消化中的复杂多肽混合物的潜力。
High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-mum wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.