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.
Highlights
In mass spectrometry-based proteomic analysis of tissues, sampling and homogenization is one of the most challenging steps, aiming at a complete release and solubilization of all proteins present in the cells and their compartments within the intact tissue before its sampling and homogenization [1]
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 nanosecond infrared laser (NIRL)
In our study we demonstrated the ablation of both murine colon and spleen tissue reproducibly with the NIRL
Summary
In mass spectrometry-based proteomic analysis of tissues, sampling and homogenization is one of the most challenging steps, aiming at a complete release and solubilization of all proteins present in the cells and their compartments within the intact tissue before its sampling and homogenization [1]. When choosing a method for homogenization, the high degree of heterogeneity of the chemical properties of proteins should be considered. This is important in the analysis of tissues, where there are many different types of cells performing specific functions in the tissue [2,3]. In the second step of homogenization, detergents or physical methods, such as osmotic shock, mechanical blending, sonication, and/or freeze/thaw treatment can be used for cell lysis [2]. Depending on the particular aim of the analysis, a combination of detergents and mechanical methods is required for the homogenization of tissues. When choosing detergents, it is essential to ensure that they are suitable for chromatography and mass spectrometry (MS) and have no interfering or suppressing effect [2]
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