Abstract
A Schwarzschild reflective objective with a numerical aperture of 0.3 and working distance of 10 cm was used for laser ablation sampling of tissue for off-line mass spectrometry. The objective focused the laser to a diameter of 5 μm and produced 10 μm ablation spots on thin ink films and tissue sections. Rat brain tissue sections 50 μm thick were ablated in transmission geometry, and the ablated material was captured in a microcentrifuge tube containing solvent. Proteins from ablated tissue sections were quantified with a Bradford assay, which indicated that approximately 300 ng of protein was captured from a 1 mm2 area of ablated tissue. Areas of tissue ranging from 0.01 to 1 mm2 were ablated and captured for bottom-up proteomics. Proteins were extracted from the captured tissue and digested for liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis for peptide and protein identification.
Highlights
Mass spectrometry is an important tool for probing biomolecular information in heterogeneous biological tissue
Initial experiments were performed to measure the diameter of the focused infrared laser (IR) laser beam and the size of laser ablation spots
The plots of transmitted laser energy and its derivative as a function of razor blade position are shown in the Supporting Information (Figure S1), and the fit of the derivative plot representing the Gaussian beam profile at the laser focal point indicates a fwhm of 5 μm
Summary
Mass spectrometry is an important tool for probing biomolecular information in heterogeneous biological tissue. The objective was used with a 3 μm optical parametric oscillator laser, and the spot size was measured by ablation of thin ink films and tissue sections. The objective was demonstrated with laser ablation sampling of rat brain tissue and proteomic characterization of the captured material. Proteins from the ablated and captured tissue were extracted and digested, and the resulting peptides were analyzed with LC−MS/MS for protein identification using a method described previously.[44] A single-pot, solid-phase-enhanced sample-preparation (SP3) approach was used to digest the captured tissue.[44,68] Ablated tissue was collected in a 300 μL microcentrifuge tube containing a 200 μL volume of 50 mM tris buffer (pH 8.5) with 1% sodium dodecyl sulfate (SDS, Sigma-Aldrich). Proteins with two or more matching (but not necessarily unique) peptides were considered identified
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