MALDI-TOF-TOF-Mass Spectrometer for mass spectrometric imaging

基质辅助激光解吸电离(MALDI)串联飞行时间质谱仪(TOF-TOF-MS)将用于组织切片的质谱学成像和生物分子(如多糖、脂类、核苷酸、多肽和蛋白质)的分子质量测定。MALDI-TOF-TOF-MS将补充现有的电喷雾电离(ESI)高分辨率串联质谱仪，因为解吸和电离机制明显不同，因此如果ESI无法检测到预期的分析物信号，将使用MALDI-TOF-TOF-MS。使用TOF-TOF质谱仪将记录分析物离子的碎片光谱，以便能够识别它们。这一功能对于质谱学成像也非常重要。后者将用于分析病理学准备的组织微阵列，用于筛选癌症标记物，分析病变组织，作为现有组织学方法的补充，并由解剖学用于分析实验模型中的组织形态。汉堡-埃彭多夫大学医学中心的核心-设施“质谱学蛋白质组学”，将使用MALDI-TOF-TOF-MS，除了上述完整离子及其片段的分子量测定外，还将用于不同形式的蛋白质组学，例如自下而上的蛋白质组学(消化蛋白质提取物，分离所得的多肽，收集MALDI靶标上的组分，用MALDI-TOF-TOF-MS分析多肽及其片段，生物信息处理和解释用于鉴定蛋白质的质谱学数据)，作为通过ESI连接到高分辨率串联质谱仪的现有液相色谱系统分析胰蛋白酶多肽的可选补充。MALDI-TOF-TOF-MS的极高速度将允许通过应用具有稳定同位素的蛋白质的代谢标记来实现蛋白质形式的相对定量蛋白质组学的新形式。将样品A和B的轻蛋白和重蛋白混合，经双向电泳分离，整个凝胶将切成约1000个方块，凝胶方块中的蛋白质被消化，得到的多肽脱盐，用MALDI-TOF-TOF-MS进行分析，并从得到的数据中确定身份和数量，由后续应用生物信息学工具确定。通过这种方法，将检测到细胞在蛋白质形式水平上的系统相关变化。MALDI-TOF-TOF-MS还将用于完整蛋白质的自上而下的分析，特别是C-末端和N-末端氨基酸序列的测定(取代Edman测序仪)。例如，这种分析对于汉堡的许多旨在对结晶蛋白质进行X射线分析的小组以及欧洲研究生院“生物分析-A4B”非常重要，该研究生院侧重于分析治疗性蛋白质的蛋白质形式。

The matrix-assisted-laser-desorption-ionization (MALDI) tandem-time-of-flight mass spectrometer (TOF-TOF-MS), which is applied for, will be used for mass spectrometric imaging of tissue sections and for determination of molecular weights of biomolecules, such as glycans, lipids, nucleotides, peptides and proteins. The MALDI-TOF-TOF-MS will supplement existing electro-spray-ionisation (ESI) high-resolution tandem mass spectrometers, since the desorption- and ionisation mechanisms are significantly different, and thus will be used, if expected signals of analytes are not detectable with ESI. With the TOF-TOF mass analyzer fragment spectra of analyte ions will be recorded, for enabling their identification. This function is also very important for mass spectrometric imaging. The later will be used for analysis of tissue-micro arrays, prepared by the Pathology, for screening for cancer markers, for analysis of diseased tissues, as an addition to existing histological methods and by the Anatomy for analysis of morphologies of tissues from experimental models. The Core-Facility "Mass Spectrometric Proteomics" of the University Medical Center Hamburg-Eppendorf, will use the MALDI-TOF-TOF-MS, beside the above mentioned determination of molecular weights of intact ions and their fragments, for different forms of proteomics, such as bottom-up proteomics (digestion of protein extracts, separation of resulting peptides, collection of the fractions on MALDI targets, analysis of the peptides and their fragments with the MALDI-TOF-TOF-MS, bioinformatic processing and interpretation of the mass spectrometric data for identification of the proteins), as an optional addition to the analysis of tryptic peptides by existing liquid chromatography systems coupled via ESI to high resolution tandem mass spectrometers. The very high speed of the MALDI-TOF-TOF-MS will allow a new form of relative quantitative proteomics of proteoforms, by applying metabolic labeling of proteins with stable isotopes. Light and heavy proteins of samples A and B will be mixed, separated by two-dimensional electrophoresis, the whole gel will be cut into approximately 1000 squares, proteins in the gel squares digested, resulting peptides desalted, analyzed by MALDI-TOF-TOF-MS and from the resulting data the identities and quantities determined by subsequent application of the bioinformatic tools. By this approach, system related changes in cells on the level of proteoforms will be detected. The MALDI-TOF-TOF-MS will be also applied for top-down analysis of intact proteins, especially for determination of C-terminal and N-terminal amino acid sequences (replacement of an Edman sequencer). This kind of analysis is e.g. important for many groups in Hamburg aiming x-ray analysis of crystalized proteins, as well as for the European graduate school "Analytics for Biologics – A4B", focusing on the analysis of proteoforms of therapeutic proteins.