Plasmonic Thermal Decomposition/Digestion of Proteins: A Rapid On-Surface Protein Digestion Technique for Mass Spectrometry Imaging

A method based on plasmon surface resonance absorption and heating was developed to perform a rapid on-surface protein thermal decomposition and digestion suitable for imaging mass spectrometry (MS) and/or profiling. This photothermal process or plasmonic thermal decomposition/digestion (plasmonic-TDD) method incorporates a continuous wave (CW) laser excitation and gold nanoparticles (Au-NPs) to induce known thermal decomposition reactions that cleave peptides and proteins specifically at the C-terminus of aspartic acid and at the N-terminus of cysteine. These thermal decomposition reactions are induced by heating a solid protein sample to temperatures between 200 and 270 °C for a short period of time (10–50 s per 200 μm segment) and are reagentless and solventless, and thus are devoid of sample product delocalization. In the plasmonic-TDD setup the sample is coated with Au-NPs and irradiated with 532 nm laser radiation to induce thermoplasmonic heating and bring about site-specific thermal decomposition on solid peptide/protein samples. In this manner the Au-NPs act as nanoheaters that result in a highly localized thermal decomposition and digestion of the protein sample that is independent of the absorption properties of the protein, making the method universally applicable to all types of proteinaceous samples (e.g., tissues or protein arrays). Several experimental variables were optimized to maximize product yield, and they include heating time, laser intensity, size of Au-NPs, and surface coverage of Au-NPs. Using optimized parameters, proof-of-principle experiments confirmed the ability of the plasmonic-TDD method to induce both C-cleavage and D-cleavage on several peptide standards and the protein lysozyme by detecting their thermal decomposition products with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The high spatial specificity of the plasmonic-TDD method was demonstrated by using a mask to digest designated sections of the sample surface with the heating laser and MALDI-MS imaging to map the resulting products. The solventless nature of the plasmonic-TDD method enabled the nonenzymatic on-surface digestion of proteins to proceed with undetectable delocalization of the resulting products from their precursor protein location. The advantages of this novel plasmonic-TDD method include short reaction times (<30 s/200 μm), compatibility with MALDI, universal sample compatibility, high spatial specificity, and localization of the digestion products. These advantages point to potential applications of this method for on-tissue protein digestion and MS-imaging/profiling for the identification of proteins, high-fidelity MS imaging of high molecular weight (>30 kDa) proteins, and the rapid analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples

Microwave Radiation Heating in Pressurized Vessels for the Rapid Extraction of Coal Samples for Broad Spectrum GC–MS Analysis

Soxhlet extraction has been successful at processing difficult to extract compounds from a variety of solid samples; however, the extraction is often time-consuming, uses large volumes of solvent, and can only process one sample at a time. This has been more evident in the sample preparation of coal and other complex geochemical samples for analysis by gas chromatography–mass spectrometry (GC–MS), where 72-h Soxhlet extractions are the norm. This study presents the development of a fast approach using a pressurized vessel system with either a hot air oven or microwave radiation heating. The techniques were tested with sub-bituminous (Powder River Range, Wyoming, U.S.A.) and bituminous (Fruitland Formation, Colorado, U.S.A.) coal samples. Performance of the pressure-vessel techniques in terms of extraction efficiency and extracted compound profiles (via GC–MS) were compared to that of a Soxhlet extraction. Overall 30–40% higher extraction efficiencies (by weight) were obtained with a 4 h hot air oven and a 20 min microwave-heating extraction in a pressurized container (using 5 mL of solvent and 1 g of coal sample) when compared to a 72 h Soxhlet extraction (using 125 mL of solvent and 25 g of coal sample). Analyses by GC–MS detected a wide range of nonpolar compounds including <i>n</i>-alkanes and diterpanes (bi-, tri-, and tetracyclic) in the sub-bituminous sample and <i>n</i>-alkanes and alkyl aromatic compounds (benzyl, naphthyl, fluorenyl, and phenanthryl) in the bituminous coal sample. The pressurized microwave heating extraction method for coal samples was found to yield extraction efficiencies that were mostly solvent independent and believed to be a result of the larger tan δ value of the coal relative to the tan δ values for the solvents tested. Advantages of the developed pressurized microwave-radiation heating method include a factor of 25 reduction in the use of solvent volume and coal sample, a 216-fold reduction of the extraction time, feasibility of parallel extractions (i.e., replication), and the ability for fully automated and safe operation of the sample preparation step