Targeted image-guided 3-D tissue sampling with nanoliter resolution for bottom-up and top-down proteomics

Proteomics of tissues using mass spectrometry-based bottom-up or top-down approaches requires sampling the area of interest, followed by homogenization. Typically, tissues consist of various cell types, each with distinct proteomes. Consequently, distinguishing areas composed of different cell types is essential to avoid mixed proteomes during sampling. In the second step, homogenizing the selected tissue breaks the compartments of the cells, releasing their molecules, including enzymes. These enzymes can alter the original composition of proteoforms, resulting in false positives and negatives. We have developed a new instrument, the 3D-miTi-LAb (3-Dimensional micro-Tissue Laser Ablation system), which allows the selection of areas of interest using microscopes and optical coherence tomography (OCT). Sampling a minimal volume of 0.5 nano-liters is achieved using a single shot of laser light with a wavelength of 2.9 µm, emitted by a nanosecond infrared laser (NIRL). During irradiation, water molecules in the tissue absorb the laser"s energy and rapidly move out, causing an explosion. This explosion converts the irradiated tissue area (80 µm diameter and 20 µm depth) into an aerosol containing all molecules previously within the tissue. Post-centrifugation, the absence of a visible pellet demonstrates effective homogenization. This is evidenced by the significantly higher yields of proteins—both in total amount and number of identified proteins—obtained through tissue sampling with NIRL compared to classical mechanical homogenization. Since NIRL-based tissue sampling is extremely fast, enzymatic degradation—typically occurring during mechanical homogenization—is minimized. Additionally, NIRL sampling is gentle, avoiding the fragmentation of labile proteins, as the laser energy is not transferred to proteins, and the exploding water molecules lose their energy via adiabatic cooling. In summary, the new 3D-miTi-LAb technology is ideal for bottom-up and top-down proteomics of tissues, offering 3-dimensional spatial resolution. It helps get closer to the original composition of proteoforms in intact tissue and minimizes mixed proteomes originating from different cell types.