On-Tissue N-Terminal Peptide Derivatizations for Enhancing Protein Identification in MALDI Mass Spectrometric Imaging Strategies
On-Tissue N-Terminal Peptide Derivatizations for Enhancing Protein Identification in MALDI Mass Spectrometric Imaging Strategies
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DOI：
10.1021/ac901043n
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发表时间：
2009-10-15
影响因子：
7.4
通讯作者：
Fournier, I.
中科院分区：
文献类型：
--
作者：
Franck, J.;El Ayed, M.;Fournier, I.
基质辅助激光解吸/电离（NMDI）是一种新的工具，可以直接从组织切片中获得各种化合物的定位，包括肽和蛋白质。尽管最近在组织中MALDI成像领域取得了重要进展，但化合物的精确识别仍需要改进。我们已经开发了N-末端化学衍生化策略，以改善蛋白质的组织鉴定，包括从头测序性能。我们首先关注磺化剂，如4-SPITC和3-SBASE。这两种衍生化被优化为直接在组织切片上进行。通过在胰蛋白酶消化肽的N-末端添加负电荷，我们能够直接从组织产生完整的y片段系列。在这些衍生化中，3-SBASE已被证明是更有效的，因为衍生基团的损失是4-SPITC的主要片段化途径之一。3-SBASE进行了优化，使衍生化反应可以使用自动微量点样装置自动进行。然后将其包括在自动化过程中，该过程包括自动化胰蛋白酶消化和基质沉积。衍生化允许通过MS 2光谱容易地解释采集，导致非常精确的鉴定以及用于从头测序的序列的容易的手动阅读。据观察，在衍生化后仅观察到甘氨酸封端的肽，这可能是由于与赖氨酸封端的肽相比，此类肽的气相碱性高。我们还观察到呈现错误切割的肽的y片段化系列中的停止。我们现在已经开始研究使用N-琥珀酰亚胺基氧羰基甲基）三（2，4，6-三甲氧基苯基）溴化鏻（TMPP）的不同衍生化。Ibis衍生化允许片段朝向一系列碎片离子定向，因此它与序列中碱性残基的存在无关。该衍生化可以在室温下进行，这极大地促进了该过程的自动化。因此，TMPP衍生化产生了适用于组织的有利的新一代衍生物。
Matrix-assisted laser desorption/ionization (NMDI) is a new tool that can acquire the localization of various compounds, including peptides and proteins, directly from tissue sections. Despite the important developments recently performed in the field of MALDI imaging in tissue, the precise identification of compounds still needs improvement. We have developed N-terminal chemical derivatization strategies to improve tissue identification of proteins, including de novo sequencing performance. We have first focused on sulfonation agents, such as 4-SPITC and 3-SBASE. These two derivatizations were optimized to be performed directly on tissue sections. By adding a negative charge at the N-terminus of a tryptic digest peptide, we were able to generate a complete y fragment series directly from the tissue. Of these derivatizations, 3-SBASE has shown to be more efficient, as loss of the derivative group is one of the major fragmentation pathways for 4-SPITC. 3-SBASE was optimized so that the derivatization reaction could be automatically performed using an automatic microspotting device. It was then included in an automatic process that included automated trypsin digestion and matrix deposition. Derivatizations allowed the acquisition to be easily interpretable by MS2 spectra, leading to very precise identification as well as easy manual reading of sequences for de novo sequencing. It was observed that only arginine-terminated peptides were observed after derivatization, likely due to the high gas-phase basicity of such peptides compared to those that are lysine-terminated. We also observed a stop in the y fragmentation series for peptides presenting a miscleavage. We have now begun to study a different derivatization using N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). Ibis derivatization allows the orientating of a fragmentation toward a series of fragment ions, and thus it is independent of the presence of basic residues in the sequence. This derivatization can be performed at room temperature, which greatly facilitates the automation of the process. The TMPP derivatization therefore yields an advantageous new generation of derivatives suited for use in tissue.