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
