On Tissue Protein Identification Improvement by N-Terminal Peptide Derivatization
On Tissue Protein Identification Improvement by N-Terminal Peptide Derivatization
复制标题
DOI：
10.1007/978-1-60761-746-4_19
复制
发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Fournier, Isabelle
中科院分区：
文献类型：
--
作者：
Franck, Julien;El Ayed, Mohamed;Fournier, Isabelle
利用MALDI-MSI发现生理阶段（如病理）的潜在标记物，是理解信号通路或为临床应用提供诊断和预后标记物的重要一步。通常，鉴定可以通过从组织中提取和蛋白质组学策略来实现。然而，这会导致信息丢失，特别是对于低丰度蛋白质或定位于组织特定区域的蛋白质。在这方面，直接在组织水平鉴定是一个有吸引力的选择。由于MALDI中的分子电荷状态较低，因此可以使用自下而上的MALDI- msi策略进行组织鉴定。使用酶如胰蛋白酶的酶消化可以在微尺度水平上进行，以产生肽集合，同时避免这些肽的离域。因此，通过蛋白质被消化肽的分子图像来成像蛋白质是可能的。这些多肽也可用于通过MS/MS检索蛋白质序列的信息，尽管使用MS/MS光谱的数据库查询或从头测序并不总是导致成功或自信的鉴定，因为组织复杂性使得PMF数据存在问题。通过提高质谱/质谱质量和简化解释，可以提高鉴定水平。这可以通过多肽的衍生化来实现。事实上，肽的衍生化导致碎片产量的增加，并将碎片定向到特定的片段离子系列。在这方面，n端化学衍生化已被证明是特别有效的。胰蛋白酶肽的n端化学衍生化已经发展到在组织消化后在组织水平上进行。特别关注的是4-硫苯基异硫氰酸酯（4- spitc）， 3-磺酸苯甲酸NHS酯（3-SBASE）和（n -丁二酰氧羰基甲基）三（2,4,6-三甲氧基苯基）溴化磷（TMPP）衍生化。这为自下而上的MALDI-MSI方法中的蛋白质鉴定提供了一个完整的策略，并为新的生物标志物鉴定开辟了道路。
Identification of potential markers of a physiological stage (e.g., pathology) discovered using MALDI-MSI is an important step in the understanding of signaling pathways or for providing sets of diagnosis and prognosis markers for clinical applications. Classically, identification can be achieved by extraction from a piece of tissue and protcomics strategies. However, this induces loss of information especially for low-abundance proteins or proteins localized to a specific region of the tissue. In this respect, identification directly at the tissue level is an attractive alternative. Because the molecular charge states in MALDI are low, on tissue identification is possible using bottom-up MALDI-MSI strategies. Enzymatic digestion using an enzyme such as trypsin can be performed at the micro-scale level to generate peptide collections while avoiding these peptides to be delocalized. It is, therefore, possible to image proteins through the molecular images of their digested peptides. These peptides can also be used to retrieve information on protein sequences by performing MS/MS, although databank interrogation or de novo sequencing using MS/MS spectra does not always lead to a successful or confident identification because on tissue complexities render PMF data problematic. Identification can be improved by increasing MS/MS spectra quality and simplifying their interpretation. This can be achieved by derivatization of peptides. In fact, derivatization of peptides leads to increases in fragmentation yields and orients fragmentations toward a specific series of fragment ions. In this respect, N-terminal chemical derivatization has proven to be particularly efficient. N-terminal chemical derivatization of tryptic peptides has been developed to be performed at the tissue level after on tissue digestion. Specific focus is given to 4-sulfophenyl isothiocyanate (4-SPITC), 3-sulfobenzoic acid NHS ester (3-SBASE), and (N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) derivatizations. This provides a complete strategy for protein identification in a bottom-up MALDI-MSI approach and opens the way for novel biomarker identification.