Thermal decomposition/digestion (TDD) of protein and its application in MALDI-MS imaging

The spatial distribution of large biomolecules, i.e. proteins, is essential to direct biological research and clinic diagnosis. MALDI-MS Imaging (MSI) is commonly used to analyze large proteins by applying an enzymatic on-surface digestion and then detecting the produced peptide fragments by MS. Although the fragments are suitable for MS analysis, the digestion process requires a long reaction time (3 hours to overnight) and induces delocalization of the products. In order to reduce the reaction time, our lab has been working on developing non-enzymatic techniques to digest proteins. In this dissertation work, a new site specific cleavage at cysteine (C) was found to be induced by thermal decomposition. In addition, a photo-induced thermal decomposition method was developed to demonstrate the potential application of TDD in MSI.;The thermal cleavage process in proteins and peptides, which happens at the N-terminus of Cys when the sample was heated with a hot gas (convective heating) in the temperature range of 200 °C to 270 °C for 30 s was studied and characterized. Using mass spectrometry, including DIP-EI-MS, MALDI-MS, MALDI-(LIFT)-MS/MS and ESI-MS/MS, the products of thermally decomposed peptides and proteins were characterized. Hydrogen sulfide was determined to be a volatile product, while non-volatile products consisted of peptides with neutral losses (water, ammonia, and hydrogen sulfide) and site-specific cleavage at Cys. Water was found to be necessary to induce the cleavage at Calpha-N bond to form the N- and C-terminal products. In addition, the N-terminal product was found to have a modification of --1 Da at the C-terminus, most likely from the amidation of the C-terminus. Meanwhile, the C-terminal product has a mass change of -33 Da via the formation of a 1,2-dione at its N-terminus. Peptides and proteins up to 66.5 kDa were successfully cleaved, with site-specificity at both Asp and Cys.;At a similar heating temperature, convincing evidence of the cleaving of disulfide bonds was collected by heating the solid proteins using the convective-heating setup at a temperature range of 220 °C-270 °C for 30 s. The non-volatile products were detected and analyzed by MALDI-MS and MALDI-MS/MS. The rate at which the disulfide bonds broke varied with heating temperatures. The thermally decomposed products were found to have the free thiol group at the amino acid Cys. In addition, the site specific cleavage at Cys occurred at a similar temperature range and as a result the C-cleavage was usually found to happen after the disulfide bond cleavage. Therefore, the TDD products detected in general were the fragments from both the C-cleavage and disulfide bonding cleavage. Several protein standards, including insulin, lysozyme and ribonuclease A, were tested to perform the thermal cleavage of disulfide bonds and successfully generate TDD fragments.;To apply these thermal decomposition cleavages on the sample preparation of MALDI-imaging, a laser induced thermal decomposition setup using gold nanoparticles (Au-NPs) was designed. In this setup, thermo-plasmonic heating of a protein surface was performed with a 532 nm laser and 50 nm diameter Au-NP's to effectively achieve site specific thermal decomposition and digestion at the amino acids Cys and Asp. Several peptides and the protein lysozyme were selected to perform these plasmonic-TDD reactions and their products detected by MALDI-MS. Consistent with the convective-TDD, cleavages from plasmonic-TDD were induced at the N-terminus of the amino acid Cys and/or at the C-terminus of the amino acid Asp. MALDI-MS imaging experiments demonstrated the spatial selectivity and specificity of the plasmonic-TDD process, illustrating the techniques potential to perform on-tissue digestions for either profile-mode or image-mode MALDI-MS tissue measurements. Conclusive evidence was obtain in the form of MALDI-MS images to indicate that the plasmonic-TDD process produces a homogenous product layer with minimal product delocalization when compared to the traditional enzymatic on-surface digestion. Overall, the advantages of the plasmonic-TDD method include short reaction time, compatibility with MS detection, high spatial specificity and no product delocalization (solventless digestion). This last advantage has direct implications in increasing the image fidelity of samples treated with on-tissue digestion and analyzed by MALDI-MS. Work is currently in progress in our laboratory to address these issues and demonstrate MS-imaging/profiling of large MW proteins directly from tissue samples.