Method development for identification of proteins at cellular resolution by on-tissue digestion and mass spectrometry imaging

利用质谱仪(MS)分析蛋白质的空间分布可以提供有关生理和病理过程的有价值的信息。到目前为止，这种方法的一个主要缺点是蛋白质信号通常不被识别，而只是报告为m/z值。另一种方法是将胰酶溶液直接涂抹在组织切片上，并对所得多肽的分布进行成像(通常为100-200微米像素大小)。我的团队已经发表了首个具有精确质量测量(3ppm均方根误差)和50微米像素大小的胰蛋白酶多肽的MS成像结果。这大大提高了蛋白质鉴定的可靠性，并使更小的组织学特征变得容易获得。拟议项目的主要目标是开发一种方法，允许在细胞维度或接近细胞维度的组织样本中可靠地鉴定和绘制蛋白质的空间图谱。胰多肽的MS成像的具体目标是：a)将空间分辨率提高到约10微米的像素大小)以确保通过准确的质量测量进行可靠的鉴定c)整合互补方法以增加识别的蛋白质数量d)针对不同组织类型定义样品制备方案和)开发高效且灵活的数据分析策略这些目标密切相关，并且需要开发分为四个工作包的全面工作流程。WP1-样品制备：胰多肽在MS成像中的空间分辨率主要不受仪器参数的限制，而主要受样品制备的限制。因此，将对胰酶和基质应用过程进行系统的研究和表征。WP2-数据采集：将调查MS成像实验的所有相关参数，以获得更高的灵敏度和更高效的MS/MS实验。用于鉴定的额外数据将通过LC-MS/MS实验和完整蛋白质的MS成像获得。WP3-验证和解释：空间分辨率将通过与组织学染色技术的比较来评估。鉴定策略将结合胰蛋白酶多肽的准确质量MS成像、LC-MS/MS数据和完整的蛋白质MS成像。数据处理选项将基于通用数据格式imzML.WP4-临床应用：方法开发基于小鼠模型组织。优化的方法将应用于有限数量的人类肿瘤活检组织，以展示方法的改进和对临床相关样本的适用性。该方法的最终目标是开发一种成像并同时识别单细胞中蛋白质的方法。这在目前的技术中是不可能的(没有先验知识)，但这是医学研究真正需要的信息，以便更好地在细胞水平上了解生理和病理过程。

Analyzing the spatial distribution of proteins by mass spectrometry (MS) imaging can provide valuable information about physiological and pathological processes. So far a major drawback of this approach is that protein signals are often not identified, but merely reported as m/z values. An alternative approach is to apply trypsin solution directly on the tissue section and image the distribution of the resulting peptides (typically at 100-200 µm pixel size). My group has published the first MS imaging results of tryptic peptides with accurate mass measurements (3 ppm root mean square error) and 50 µm pixel size. This significantly increased the reliability of protein identification and made smaller histological features accessible.The main objective of the proposed project is to develop a method that allows the reliable identification and spatial mapping of proteins in tissue samples at or close to cellular dimensions. Specific goals for MS imaging of tryptic peptides are:a) to increase spatial resolution to a pixel size of about 10 µmb) to ensure reliable identification by accurate mass measurementsc) to integrate complementary methods in order to increase number of identified proteinsd) to define sample preparation protocols for different tissue types ande) to develop efficient and flexible data analysis strategiesThese goals are closely related and require the development of a comprehensive workflow which is divided in four workpackages.WP1 - Sample preparation: Spatial resolution in MS imaging of tryptic peptides is not primarily limited by instrumental parameters, but by sample preparation. Therefore a systematic investigation and characterization of the trypsin and matrix application process will be performed.WP2 - Data acquisition: All relevant parameters of MS imaging experiments will be investigated in order to obtain higher sensitivity and more efficient MS/MS experiments. Additional data for identification will be obtained by LC-MS/MS experiments and MS imaging of intact proteins.WP3 - Validation and interpretation: Spatial resolution will be evaluated by comparison to histological staining techniques. Identification strategies will combine accurate mass MS imaging of tryptic peptides, LC-MS/MS data and intact protein MS imaging. Data processing options will be developed based on the common data format imzML.WP4 - Clinical application: Method development is based on mouse model tissue. The optimized methods will be applied to a limited number of human tumor biopsies in order to demonstrate methodological improvements and applicability for clinically relevant samples.The ultimate goal of this approach is to develop a method to image and simultaneously identify proteins in single cells. This is not possible (without prior knowledge) with current techniques, but it is the information that medical researches truly require in order to better understand physiological and pathological processes on a cellular level.