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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.

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.

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.

Significant changes of glycan structures are observed in humans if diseases like cancer, arthritis or inflammation are present. Thus, interest in biomarkers based on glycan structures has rapidly emerged in recent years and monitoring disease specific changes of glycosylation and their quantification is of great interest. Mass spectrometry is most commonly used to characterize and quantify glycopeptides and glycans liberated from the glycoprotein of interest. However, ionization properties of glycopeptides can strongly depend on their composition and can therefore lead to intensities that do not reflect the actual proportions present in the intact glycoprotein. Here we show that an increase in the length of the peptide can lead to a more accurate determination and quantification of the glycans. The four glycosylation sites of human serum ceruloplasmin from 17 different individuals were analyzed using glycopeptides of varying peptide lengths, obtained by action of different proteases and by limited digestion. In most cases, highly sialylated compositions showed an increased relative abundance with increasing peptide length. We observed a relative increase of triantennary glycans of up to a factor of three and, even more, MS peaks corresponding to tetraantennary compositions on ceruloplasmin at glycosite 137N in all 17 samples, which we did not detect using a bottom up approach. The data presented here leads to the conclusion that a middle down - or when possible a top down - approach is favorable for qualitative and quantitative analysis of the glycosylation of glycoproteins.