Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.
Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.
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用于通过 MALDI-TOF MS 和 MS/MS 分析肽的香豆素标签。
DOI：
10.1021/ac048375g
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发表时间：
2005
影响因子：
7.4
通讯作者：
Karger,BarryL
中科院分区：
文献类型：
--
作者：
Pashkova,Anna;Chen,Hsuan-Shen;Rejtar,Tomas;Zang,Xin;Giese,Roger;Andreev,Victor;Moskovets,Eugene;Karger,BarryL
本研究的目标是开发N末端标签，以提高使用高通量MALDI-TOF/TOF MS进行肽识别的能力。研究的第一部分重点关注衍生化对肽的MALDI-TOF MS信号强度的影响。在第二部分中，各种衍生化方法，提高MALDI-TOF/TOF MS中的肽片段化效率进行了探讨。我们表明，永久性阳离子标签，而显着提高信号强度的MS模式，导致严重抑制MS/MS碎片，使这些标签不适合高通量MALDI-TOF/TOF MS分析。在本工作中，发现用Alexa Fluor 350（一种含有磺基的香豆素标签）标记，沿着赖氨酸的ε-氨基的胍基化，可以增强肽的单分子片段化，形成高强度的γ-离子系列，而MS模式中的肽强度没有受到严重影响。来自大肠杆菌SCX级分的胰蛋白酶肽的LC-MALDI-TOF/TOF MS分析。大肠杆菌裂解物显示，作为标记的结果，肽评分提高，肽的总数加倍，鉴定的蛋白质的数量增加30%。此外，通过组合来自天然样品和标记样品的数据，增加了正确鉴定的置信度，因为许多蛋白质由天然和标记数据集中的不同肽鉴定。此外，发现衍生化不会损害肽的色谱行为。所有这些因素表明，Alexa Fluor 350标记是一种有前途的高通量LC-MALDI-TOF/TOF MS分析蛋白质组样品的方法。
The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of ε-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of anE. colilysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.