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.

For investigating the molecular physiology and pathophysiology in organs, the most exact data should be obtained; if not, organ-specific cell lines are analyzed, or the whole organ is homogenized, followed by the analysis of its biomolecules. However, if the morphological organization of the organ can be addressed, then, in the best case, the composition of molecules in single cells of the target organ can be analyzed. Laser capture microdissection (LCM) is a technique which enables the selection of specific cells of a tissue for further analysis of their molecules. However, LCM is a time-consuming two-dimensional technique, and optimal results are only obtained if the tissue is fixed, e.g., by formalin. Especially for proteome analysis, formalin fixation reduced the number of identifiable proteins, and this is an additional drawback. Recently, it was demonstrated that sampling of fresh-frozen (non-fixed) tissue with an infrared-laser is giving higher yields with respect to the absolute protein amount and number of identifiable proteins than conventional mechanical homogenization of tissues. In this study, the applicability of the infrared laser tissue sampling for the proteome analysis of different cell layers of murine intestine was investigated, using LC–MS/MS-based differential quantitative bottom-up proteomics. By laser ablation, eight consecutive layers of colon tissue were obtained and analyzed. However, a clear distinguishability of protein profiles between ascending, descending, and transversal colon was made, and we identified the different intestinal-cell-layer proteins, which are cell-specific, as confirmed by data from the Human Protein Atlas. Thus, for the first time, sampling directly from intact fresh-frozen tissue with three-dimensional resolution is giving access to the different proteomes of different cell layers of colon tissue.

Common workflows in bottom-up proteomics require homogenization of tissue samples to gain access to the biomolecules within the cells. The homogenized tissue samples often contain many different cell types, thereby representing an average of the natural proteome composition, and rare cell types are not sufficiently represented. To overcome this problem, small-volume sampling and spatial resolution are needed to maintain a better representation of the sample composition and their proteome signatures. Using nanosecond infrared laser ablation, the region of interest can be targeted in a three-dimensional (3D) fashion, whereby the spatial information is maintained during the simultaneous process of sampling and homogenization. In this study, we ablated 40 μm thick consecutive layers directly from the scalp through the cortex of embryonic mouse heads and analyzed them by subsequent bottom-up proteomics. Extra- and intracranial ablated layers showed distinct proteome profiles comprising expected cell-specific proteins. Additionally, known cortex markers like SOX2, KI67, NESTIN, and MAP2 showed a layer-specific spatial protein abundance distribution. We propose potential new marker proteins for cortex layers, such as MTA1 and NMRAL1. The obtained data confirm that the new 3D tissue sampling and homogenization method is well suited for investigating the spatial proteome signature of tissue samples in a layerwise manner. Characterization of the proteome composition of embryonic skin and bone structures, meninges, and cortex lamination in situ enables a better understanding of molecular mechanisms of development during embryogenesis and disease pathogenesis.

Radiotherapy (RT) is a central treatment for prostate cancer (PCa), relying on the induction of DNA double-strand breaks (DSBs). Tumor ability to repair these breaks limits RT efficacy, making DSB repair inhibitors potential radiosensitizers. However, many of these inhibitors lack tumor specificity and harm normal cells. Therefore, tumor-specific radiosensitization strategies are critically needed for PCa. Approximately 50% of PCa cases harbor the TMPRSS2-ERG gene fusion, leading to overexpression of the ERG transcription factor (ERG ⁺ ). In this study, we demonstrate that ERG ⁺ tumors shift DSB repair toward the poly(ADP-ribose) polymerase 1–dependent end-joining (PARP1-EJ) pathway. Proteomic and Western blot analyses revealed elevated PARP1, XRCC1, and LIG3 levels in ERG ⁺ cells. Notably, PARP inhibition with olaparib increased residual γH2AX/53BP1 foci postirradiation in ERG ⁺ cells, indicating enhanced radiosensitization. In tissue slice cultures (TSCs) from 53 tumors of patients with high-risk PCa, olaparib selectively increased γH2AX/53BP1 foci selectively in ERG ⁺ samples. ERG ⁺ patient–derived organoids also showed significantly delayed growth when treated with olaparib plus RT, compared with either treatment alone. Interestingly, ERG-negative cells within ERG ⁺ TSCs were similarly radiosensitized by olaparib, likely through bystander effect, with residual 53BP1 foci levels comparable to those in ERG ⁺ cells. This was confirmed by medium exchange experiments. These findings suggest that ERG expression promotes dependency on the PARP1-EJ pathway, rendering ERG ⁺ PCa more susceptible to PARP inhibition. This supports combining PARP inhibitors with RT for tumor-selective radiosensitization in ERG ⁺ patients.

Ultrashort pulse infrared lasers can simultaneously sample and homogenize biological tissue using desorption by impulsive vibrational excitation (DIVE). With growing attention on alterations in lipid metabolism in malignant disease, mass spectrometry (MS)-based lipidomic analysis has become an emerging topic in cancer research. In this pilot study, we investigated the feasibility of tissue sampling with a nanosecond infrared laser (NIRL) for the subsequent lipidomic analysis of oropharyngeal tissues, and its potential to discriminate oropharyngeal squamous cell carcinoma (OPSCC) from non-tumorous oropharyngeal tissue. Eleven fresh frozen oropharyngeal tissue samples were ablated. The produced aerosols were collected by a glass fiber filter, and the lipidomes were analyzed with mass spectrometry. Data was evaluated by principal component analysis and Welch’s t-tests. Lipid profiles comprised 13 lipid classes and up to 755 lipid species. We found significant inter- and intrapatient alterations in lipid profiles for tumor and non-tumor samples (p-value < 0.05, two-fold difference). Thus, NIRL tissue sampling with consecutive MS lipidomic analysis is a feasible and promising approach for the differentiation of OPSCC and non-tumorous oropharyngeal tissue and may provide new insights into lipid composition alterations in OPSCC.

A nanosecond infrared laser (NIRL) was investigated in cutting dental roots. The focus of the investigation was defining the preparation accuracy and registration of thermal effects during laser application. Ten teeth were processed in the root area using a NIRL in several horizontal, parallel incisions to achieve tooth root ablation as in an apicoectomy. Temperature change was monitored during ablation and the quality of the cutting edges in the roots were studied by means of micro-CT, optical coherence tomography, and histology of decalcified and undecalcified specimens. NIRL produced clearly defined cut surfaces in dental hard tissues. The automated guidance of the laser beam created regular, narrow dentin defects that tapered in a V-shape towards the ablation plane. A biologically significant increase in the temperature of the object and its surroundings did not occur during the laser application. Thermal dentin damage was not detected in histological preparations of treated teeth. Defined areas of the tooth root may be ablated using a NIRL. For clinical translation of NIRL in apicoectomy, it would be necessary to increase energy delivered to hard tissue and develop beam application facilitating beam steering for oral treatment.