Tissue Sampling and Homogenization in the Sub-Microliter Scale with a Nanosecond Infrared Laser (NIRL) for Mass Spectrometric Proteomics

Abstract

It was recently shown that ultrashort pulse infrared (IR) lasers, operating at the wavelength of the OH vibration stretching band of water, are highly efficient for sampling and homogenizing biological tissue. In this study we utilized a tunable nanosecond infrared laser (NIRL) for tissue sampling and homogenization with subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for mass spectrometric proteomics. For the first time, laser sampling was performed with murine spleen and colon tissue. An ablation volume of 1.1 × 1.1 × 0.4 mm³ (approximately 0.5 µL) was determined with optical coherence tomography (OCT). The results of bottom-up proteomics revealed proteins with significant abundance differences for both tissue types, which are in accordance with the corresponding data of the Human Protein Atlas. The results demonstrate that tissue sampling and homogenization of small tissue volumes less than 1 µL for subsequent mass spectrometric proteomics is feasible with a NIRL.

Tissue Sampling and Homogenization with NIRL Enables Spatially Resolved Cell Layer Specific Proteomic Analysis of the Murine Intestine

“…In comparison to our most recent study [ 26 ], where 1617 proteins were identified from murine colon tissue, using a similar setup, we demonstrated a significant increase in the number of identified proteins (3053) for a comparable ablation volume (approximately 0.6 µL). This increased efficiency can be explained by direct trapping of the aerosol on a glass slide above the tissue sample and reducing the scanning area for the laser ablation by shifting of sample repeatedly.…”

“…Besides PIRL [ 21 , 22 , 23 ] and microsecond infrared laser (MIRL) [ 23 ]. especially nanosecond infrared lasers (NIRL) with a pulse duration of about 7 ns, have been successfully utilized for tissue sampling prior to proteome analysis [ 24 , 25 , 26 ]. In the recent study by our group, Hahn et al [ 26 ] revealed the clear distinguishability of murine colon and spleen tissue, based on >1000 identified proteins after collecting NIRL-induced tissue aerosols as condensates on glass cover slips, followed by bottom-up proteomics.…”

Infrared Laser Ablation Microsampling with a Reflective Objective

“…38 − 42 IR laser ablation and capture has been demonstrated for proteomic analysis from millimeter-sized tissue regions with a sampling spot size of approximately 200 μm. 43 − 45 …”

“…Mid-IR lasers with wavelengths ∼3 μm are efficient for ablation of biological tissue because of wavelength overlap with OH vibrational absorption of water molecules . Laser heating of the water in the tissue leads to a rapid volumetric phase change and ablation of the irradiated region, resulting in removal of the disrupted tissue; no significant biomolecule fragmentation has been reported. − IR laser ablation and capture has been demonstrated for proteomic analysis from millimeter-sized tissue regions with a sampling spot size of approximately 200 μm. − …”

Proteomic Landscape of Human Spermatozoa: Optimized Extraction Method and Application

“…The commonly used buffers for protein extraction are SDS and UA buffer, which readily solubilize most proteins in different clinical samples [36,37]. Commonly used extraction methods include ultrasonic instruments and homogenate instruments, which promote sample fragmentation and protein dissolution [38,39]. The selection of an appropriate buffer and extraction instrument will help us extract adequate proteins for precise qualitative and quantitative analyses in clinical samples.…”