Linear copolymers or terpolymers with appropriate chemical composition (wt% of each monomer), sequence of the monomeric units (block or random) and nature of the monomer (styrene or methacrylate) will be synthesized by NMRP or Atom Transfer Radical Polymerization (ATRP). These polymers will be capable of positive, or negative, or both modes analysis in MALDI. Initially, diblock copolymers or random copolymers of methacrylate-pyrene monomer (or styrene pyrene monomer), consecutively, or together, with acrylic acid will be synthesized by the above controlled/living radical polymerizations. Following the same synthetic protocol, diblock copolymers or random copolymers of methacrylate-pyrene monomer (or styrene-pyrene monomer), consecutively, or together, with diethylamino ethyl methacrylate will be synthesized by controlled/living radical polymerizations. As described above, molecular characterization with SEC, 1H-NMR and MALDI will be carried out for each of the synthesized polymer.

Synthesis of linear random terpolypeptides capable of both positive and negative mode, with increased solubility in common organic solvents, high absorbance at 337 nm, low molecular weight distribution and molecular weights ranging between 5,000 Da and 15,000 Da. The synthesis of the terpolypeptides will involve the ring-opening polymerization of the N-carboxy anhydrides of the protected amino acids. For the monomer of L-lysine, BOC-protection will be used, while for the L-glutamic acid the benzyl ester will be used as the protective group. The monomers will be synthesized by reaction of the protected amino acids with triphosgene.

The development of complementary methods that will provide high-throughput, comprehensive and unambiguous identification, and quantification of a wide range of biologically relevant  LMWCs, utilizing state of the art MALDI instrumentation. For each combination of matrix and groups of analytes, crucial instrumental parameters (MALDI, TIMS parameters) will be optimized for each detection mode improving throughput and depth of instrumentation. Improvements in sample preparation will be made to ensure reliable interpretation of data. Statistical workflows and machine learning algorithms will be implemented to handle the large datasets being produced. Molecular information will be implemented into molecular pathway databases for the complete characterization of LMW content in samples of interest and supervised and unsupervised prediction models will be applied for discrimination purposes.

Aiming at the insurance of scientific correctness, a validation study will be performed to assure the overall performance of each developed analytical methodology. Numerous relevant overall performance indicators will be tested, including selectivity, specificity, accuracy, precision, linearity, range, limit of detection (LOD) and quantification (LOQ) of the analytes, ruggedness, and robustness.

MALDI-TIMS-HRMS based Imaging analyses of the aforementioned samples (Task 3.1) will be performed, aiming to cover a wide range of expressed (endogenous) molecules such as metabolites, lipids, and peptides. Sophisticated data processing tools will be used that allows the reconstruction, visualization, and statistical analysis of 2D and 3D Imaging datasets. Spatially significant expression of endogenous molecules will be evaluated. Regiospecific molecular differences will be identified in a multi-omic perspective, aiming to define tissue regions of interest (ROI). Regionally specific information will be combined with deep metabolomic, lipidomic and proteomic coverage as well as xenometabolome assessment (Task 3.1) for biomarker discovery and molecular characterization of the induced toxicity.

Brightfield microscopy of stained tissues (hematoxylin and eosin stain) will be performed to obtain histological data for the exposed zebrafish. The thousands of ion images produced by the IMS experiments (Taks 3.1 & 3.2) will be integrated with histologically stained sections providing molecular context to histological analysis.