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Deep-ultraviolet laser ablation with a pulsed 193 nm ArF excimer laser was used to remove localized regions from tissue sections from which proteins were extracted for spatially resolved proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The ability to capture intact proteins by ablation at 193 nm wavelength was verified by matrix-assisted laser desorption ionization (MALDI) of the protein standard bovine serum albumin (BSA), which showed that BSA was ablated and captured without fragmentation. A Bradford assay of the ablated and captured proteins indicated 90% efficiency for transfer of the intact protein at a laser fluence of 3 kJ/m 2 . Rat brain tissue sections mounted on quartz microscope slides and ablated in transmission mode yielded 2 μg protein per mm 2 as quantified by the Bradford assay. Tissue areas ranging from 0.06 mm 2 to 1 mm 2 were ablated and the ejected material was collected for proteomic analysis. Extracted proteins were digested and the resulting peptides were analyzed by LC-MS/MS. The proteins extracted from the ablated areas were identified and the average number of identified proteins ranged from 85 in the 0.06 mm 2 area to 2400 in the 1 mm 2 area of a 50 μm thick tissue. In comparison to infrared laser ablation of equivalent sampled areas, both the protein mass and number of proteins identified using DUV laser ablation sampling were approximately four times larger., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

RNA was obtained from discrete locations of frozen rat brain tissue sections through infrared (IR) laser ablation using a 3-μm wavelength in transmission geometry. The ablated plume was captured in a microcentrifuge tube containing RNAse-free buffer and processed using a commercial RNA purification kit. RNA transfer efficiency and integrity were evaluated based on automated electrophoresis in microfluidic chips. Reproducible IR-laser ablation of intact RNA was demonstrated with purified RNA at laser fluences of 3-5 kJ/m 2 (72 ± 12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m 2 (79 ± 14% transfer efficiency); laser energies were attenuated ∼20% by the soda-lime glass slides used to support the samples. RNA integrity from tissue ablation was >90% of its original RIN value (∼7) and the purified RNA was sufficiently intact for conversion to cDNA and subsequent qPCR assay., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Infrared laser ablation microsampling was used with data-dependent acquisition (DDA) and ion mobility-enhanced data-independent acquisition (HDMS E ) for mass spectrometry based bottom-up proteomics analysis of rat brain tissue. Results from HDMS E and DDA analyses of the 12 laser ablation sampled tissue sections showed that HDMS E consistently identified approximately seven times more peptides and four times more proteins than DDA. To evaluate the impact of ultra-performance liquid chromatography (UPLC) peak congestion on HDMS E and DDA analysis, whole tissue digests from rat brain were analyzed at six different UPLC separation times. Analogous to results from laser ablated samples, HDMS E analyses of whole tissue digests yielded about four times more proteins identified than DDA for all six UPLC separation times., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Infrared (IR) laser ablation at 3 μm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ± 8% at laser fluences between 10 and 30 kJ/m 2 . Trypsin retained 37 ± 2% of its activity and catalase retained 50 ± 7%. The activity of catalase from tissue was tested using three consecutive 50 μm thick rat brain sections. Two 4 mm 2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A systematic method for evaluation of MALDI profiling and imaging was developed and applied to the use of three surfactants, sodium dodecyl sulfate (SDS), Triton X-100, and Tween 20, on rat brain tissue. For profiling studies, mass spectra were acquired from regular arrays of spots with manually deposited surfactant and matrix. The studies recorded the total number of peaks in the mass spectra from 2 to 20 kDa and compared the number of peaks and peak intensities with and without surfactant. It was found that SDS decreases the total number of peaks at all concentrations but does lead to an increase in the number of peaks below 5 kDa. Triton X-100 at 0.05% concentration yielded the highest number of peaks and highest number of new peaks, with the best results above 5 kDa. Correlation of the increase in signal with the estimated hydrophobicity suggests that Triton X-100 improves mass spectrometry quality through an increase in the intensity of hydrophobic protein peaks. Tween 20 provided good performance at 0.05% concentration across all mass ranges. For imaging studies, multiple images were obtained and the integrated intensity ratio for images obtained with and without surfactant was compared for 10 selected peaks. It was found that SDS tends to degrade imaging performance whereas Triton X-100 and Tween 20 improved performance compared to no surfactant, especially above 7 kDa., (Copyright © 2017 Elsevier B.V. All rights reserved.)