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

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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.