Laser ablation sample transfer for localized LC‐MS/MS proteomic analysis of tissue

Abstract

We have developed a mid-infrared laser ablation sampling technique for nano-flow liquid chromatography coupled with tandem mass spectrometry proteomic profiling of discrete regions from biological samples. Laser ablation performed in transmission geometry was used to transfer material from 50-µm thick tissue sections mounted on a glass microscope slide to a capturing solvent. Captured samples were processed using filter-aided sample preparation and enzymatically digested to produce tryptic peptides for data-dependent analysis with an ion trap mass spectrometer. Comparison with ultraviolet laser capture microdissection from neighboring regions on the same tissue section revealed that infrared laser ablation transfer has higher reproducibility between samples from different consecutive sections. Both techniques allowed for proteomics investigation of different organelles without the addition of surfactants.

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics

“…Matrix removal was accomplished by washing tissue sections with 70% ethanol for 1 minute followed by rehydration via submersion in water for 10 minutes. The mid‐IR laser ablation system has been previously described . Briefly, a tunable wavelength (λ = 2940 nm) pulsed (20 Hz) nanosecond (7‐ns temporal width) IR optical parametric oscillator (IR Opolette; OPOTEK, Carlsbad, CA, USA) was used to irradiate samples using a transmission geometry.…”

“…Single‐pot solid‐phase‐enhanced sample preparation (SP3) was used for tissue digestion and filter aided sample preparation (FASP) . Further details can be found in Supplementary Material Figure S2.…”

“…Laser ablation and capture can be used to remove material from tissue sections by converting it to small particles that are ejected from the tissue into a capture vial or analysis system. Laser ablation uses a pulsed UV, visible, or IR laser to irradiate the regions of tissue section in transmission (back side irradiation) or reflection (front‐side radiation) geometry . The mid‐IR wavelength of 2.94 μm overlaps with OH vibrational absorption of water as well as molecules containing a hydroxyl group and is efficient at biomolecule ablation without fragmentation .…”

“…The mid‐IR wavelength of 2.94 μm overlaps with OH vibrational absorption of water as well as molecules containing a hydroxyl group and is efficient at biomolecule ablation without fragmentation . Mid‐IR laser ablation has been used for ablation capture and offline analysis of proteins, peptides, enzyme, DNA, and RNA while preserving their molecular integrity.…”

Systematic assessment of surfactants for matrix-assisted laser desorption/ionization mass spectrometry imaging

“…The lateral resolution is limited by the laser beam diameter, the raster step size, and the diffusion of the biomolecules due to matrix addition [11], The mass range is determined by mass analyzer and detector [12] as well as the method of sample preparation [13]. Digestion of proteins directly on tissue [14,15] or after extraction from it [16][17][18] can be used to extend the effective mass range and aid in compound identification.…”