Mass spectrometry for neurosurgery: Intraoperative support in decision-making.

Ambient ionization mass spectrometry was proved to be a powerful tool for oncological surgery. Still, it remains a translational technique on the way from laboratory to clinic. Brain surgery is the most sensitive to resection accuracy field since the balance between completeness of resection and minimization of nerve fiber damage determines patient outcome and quality of life. In this review, we summarize efforts made to develop various intraoperative support techniques for oncological neurosurgery and discuss difficulties arising on the way to clinical implementation of mass spectrometry-guided brain surgery.

Modern machine-learning applications in ambient ionization mass spectrometry.

This article provides a comprehensive overview of the applications of methods of machine learning (ML) and artificial intelligence (AI) in ambient ionization mass spectrometry (AIMS). AIMS has emerged as a powerful analytical tool in recent years, allowing for rapid and sensitive analysis of various samples without the need for extensive sample preparation. The integration of ML/AI algorithms with AIMS has further expanded its capabilities, enabling enhanced data analysis. This review discusses ML/AI algorithms applicable to the AIMS data and highlights the key advancements and potential benefits of utilizing ML/AI in the field of mass spectrometry, with a focus on the AIMS community.

Molecular Biomarkers in Cholangiocarcinoma: Focus on Bile.

Hepatobiliary system cancers have demonstrated an increasing incidence rate in the past years. Without the presence of early symptoms, the majority of such cancers manifest with a set of similar symptoms, such as cholestasis resulting in posthepatic icterus. Differential diagnosis of hepatobiliary cancers is required for the therapy selection, however, the similarity of the symptoms complicates diagnostics. Thus, the search for molecular markers is of high interest for such patients. Cholangiocarcinoma (CCA) is characterized by a poor prognosis due to a low resectability rate, which occurs because this disease is frequently beyond the limits of surgical therapy at the time of diagnosis. The CCA is diagnosed by the combination of clinical/biochemical features, radiological methods, and non-specific serum tumor biomarkers, although invasive examination is still needed. The main disadvantage is limited specificity and sensitivity, which complicates early diagnostics. Therefore, prognostic and predictive biomarkers are still lacking and urgently needed for early diagnosis. In contrast to serum, bile is more accessible to identify biliary disease due to its simpler composition. Moreover, bile can contain higher concentrations of tumor biomarkers due to its direct contact with the tumor. It is known that the composition of the main bile component - bile acids, may vary during different diseases of the biliary tract. This review summarizes the recent developments in the current research on the diagnostic biomarkers for CCA in serum and bile and provides an overview of the methods of bile acids analysis.

Incorporation of a Disposable ESI Emitter into Inline Cartridge Extraction Mass Spectrometry Improves Throughput and Spectra Stability.

Rapid and reliable methods for detecting tumor margins are crucial for neuro-oncology. Several mass spectrometry-based methods have been recently proposed to address this problem. Inline Cartridge Extraction (ICE) demonstrates the potential for clinical application, based on ex-vivo analysis of dissected tissues, but requires time-consuming steps to avoid cross-contamination. In this work, a method of incorporating a disposable electrospray emitter into the ICE cartridge by PEEK sleeves melting is developed. It reduces total analysis time and improves throughput. The proposed setup also improves the robustness of the ICE molecular profiling as demonstrated with human glial tumor samples in that stability and reproducibility of the spectra were increased.

Quantitative Assessment of Serine-8 Phosphorylated ß-Amyloid Using MALDI-TOF Mass Spectrometry.

The study of the molecular mechanisms of the pathogenesis of Alzheimer's disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of ß-amyloid (Aß) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aß) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aß based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aß was estimated in the total Aß-pool with a detection limit of 1 fmol for pSer8-Aß (1-16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples.

Determination of Brain Tissue Samples Storage Conditions for Reproducible Intraoperative Lipid Profiling.

Ex-vivo molecular profiling has recently emerged as a promising method for intraoperative tissue identification, especially in neurosurgery. The short-term storage of resected samples at room temperature is proposed to have negligible influence on the lipid molecular profiles. However, a detailed investigation of short-term molecular profile stability is required to implement molecular profiling in a clinic. This study evaluates the effect of storage media, temperature, and washing solution to determine conditions that provide stable and reproducible molecular profiles, with the help of ambient ionization mass spectrometry using rat cerebral cortex as model brain tissue samples. Utilizing normal saline for sample storage and washing media shows a positive effect on the reproducibility of the spectra; however, the refrigeration shows a negligible effect on the spectral similarity. Thus, it was demonstrated that up to hour-long storage in normal saline, even at room temperature, ensures the acquisition of representative molecular profiles using ambient ionization mass spectrometry.

Spherical Sampler Probes Enhance the Robustness of Ambient Ionization Mass Spectrometry for Rapid Drugs Screening.

Ambient ionization mass spectrometry has become one of the most promising approaches for rapid and high-throughput screening of small molecules in complex biological matrices for emergency medicine, forensics, and food and agriculture applications. The simple procedures for sample collection and ionization without additional pretreatment are vital in these fields. Many efforts have been devoted to modifying various ambient ionization techniques to simplify the procedures and improve the robustness and sensitivity of the methods. Here, we demonstrate the implementation of rigid spherical sampler probes to improve the robustness of touch spray ionization mass spectrometry. The sphericity of the probes increases the stability of the cone-jet mode of electrospray, reduces the requirements for fine positioning of a sampler in the ion source, and decreases the possibility of corona discharge occurrence. The utilization of spherical sampler probes allows fast, non-invasive sampling, followed by rapid analysis for various drugs of different chemical classes in complex biological matrices, such as the whole blood or sebum collected from the skin surface. The linearity of the analytical signal response from drug concentration confirms the possibility of creating a simple semiquantitative method for small molecules monitoring using spherical sampler probes.

Cell-Population Dynamics in Diffuse Gliomas during Gliomagenesis and Its Impact on Patient Survival.

Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations' content in diffuse gliomas of different molecular profiles and grades, considering the cell populations' functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells' proportion and mesenchymal cell populations' appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells' hypoxia response programs' activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs' activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas' pathogenetic tracing up to the practical plane.

Rapid estimation of tumor cell percentage in brain tissue biopsy samples using inline cartridge extraction mass spectrometry.

Tumor cell percentage (TCP) is an essential characteristic of biopsy samples that directly affects the sensitivity of molecular testing in clinical practice. Apart from clarifying diagnoses, rapid evaluation of TCP combined with various neuronavigation systems can be used to support decision making in neurosurgery. It is known that ambient mass spectrometry makes it possible to rapidly distinguish healthy from malignant tissues. In connection with this, here we demonstrate the possibility of using non-imaging ambient mass spectrometry to evaluate TCP in glial tumor tissues with a high degree of confidence. Molecular profiles of histologically annotated human glioblastoma tissue samples were obtained using the inline cartridge extraction ambient mass spectrometry approach. XGBoost regressors were trained to evaluate tumor cell percentage. Using cross-validation, it was estimated that the TCP was determined by the regressors with a precision of approximately 90% using only low-resolution data. This result demonstrates that ambient mass spectrometry provides an accurate method todetermine TCP in dissected tissues even without implementing mass spectrometry imaging. The application of such techniques offers the possibility to automate routine tissue screening and TCP evaluation to boost the throughput of pathology laboratories. Rapid estimation of tumor cell percentage during neurosurgery.

The Dynamics of ß-Amyloid Proteoforms Accumulation in the Brain of a 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Neuropathologically, AD is characterized by the deposition of a 39- to 42-amino acid long ß-amyloid (Aß) peptide in the form of senile plaques. Several post-translational modifications (PTMs) in the N-terminal domain have been shown to increase the aggregation and cytotoxicity of Aß, and specific Aß proteoforms (e.g., Aß with isomerized D7 (isoD7-Aß)) are abundant in the senile plaques of AD patients. Animal models are indispensable tools for the study of disease pathogenesis, as well as preclinical testing. In the presented work, the accumulation dynamics of Aß proteoforms in the brain of one of the most widely used amyloid-based mouse models (the 5xFAD line) was monitored. Mass spectrometry (MS) approaches, based on ion mobility separation and the characteristic fragment ion formation, were applied. The results indicated a gradual increase in the Aß fraction of isoD7-Aß, starting from approximately 8% at 7 months to approximately 30% by 23 months of age. Other specific PTMs, in particular, pyroglutamylation, deamidation, and oxidation, as well as phosphorylation, were also monitored. The results for mice of different ages demonstrated that the accumulation of Aß proteoforms correlate with the formation of Aß deposits. Although the mouse model cannot be a complete analogue of the processes occurring in the human brain in AD, and several of the observed parameters differ significantly from human values supposedly due to the limited lifespan of the model animals, this dynamic study provides evidence on at least one of the possible mechanisms that can trigger amyloidosis in AD, i.e., the hypothesis on the relationship between the accumulation of isoD7-Aß and the progression of AD-like pathology.

Assessment of variation of inline cartridge extraction mass spectra.

Recently, mass-spectrometry methods show its utility in tumor boundary location. The effect of differences between research and clinical protocols such as low- and high-resolution measurements and sample storage have to be understood and taken into account to transfer methods from bench to bedside. In this study, we demonstrate a simple way to compare mass spectra obtained by different experimental protocols, assess its quality, and check for the presence of outliers and batch effect in the dataset. We compare the mass spectra of both fresh and frozen-thawed astrocytic brain tumor samples obtained with the inline cartridge extraction prior to electrospray ionization. Our results reveal the importance of both positive and negative ion mode mass spectrometry for getting reliable information about sample diversity. We show that positive mode highlights the difference between protocols of mass spectra measurement, such as fresh and frozen-thawed samples, whereas negative mode better characterizes the histological difference between samples. We also show how the use of similarity spectrum matrix helps to identify the proper choice of the measurement parameters, so data collection would be kept reliable, and analysis would be correct and meaningful.

Relative Quantitation of Beta-Amyloid Peptide Isomers with Simultaneous Isomerization of Multiple Aspartic Acid Residues by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry can be used for rapid quantitation of peptides with various post-translational modifications (PTM), even if they do not shift the mass of the native peptide. Previously, it was shown that MALDI-TOF MS can be used for quantitation of isoD7 beta-amyloid 1-42 peptide. On the basis of the differences in the collision-induced dissociation fragmentation pattern of native Aß, isoD7 Aß, isoD23 Aß, and isoD7_23 peptide (a di-isomerized peptide with both isomerization of D7 and D23 residues), we developed a MALDI-TOF-based method for simultaneous quantitation of all of these isoforms. Using multivariate regression for analysis of fragment MS data, the method allows the determination of the molar fractions of all of these isoforms with up to 16% error for mixtures with 2 pmol total amount of the beta-amyloid peptide.

Inline cartridge extraction for rapid brain tumor tissue identification by molecular profiling.

The development of perspective diagnostic techniques in medicine requires efficient high-throughput biological sample analysis methods. Here, we present an inline cartridge extraction that facilitates the screening rate of mass spectrometry shotgun lipidomic analysis of tissue samples. We illustrate the method by its application to tumor tissue identification in neurosurgery. In perspective, this high-performance method provides new possibilities for the investigation of cancer pathogenesis and metabolic disorders.

Probabilistic model applied to ion abundances in product-ion spectra: quantitative analysis of aspartic acid isomerization in peptides.

Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.

Evaluation of MALDI-TOF/TOF Mass Spectrometry Approach for Quantitative Determination of Aspartate Residue Isomerization in the Amyloid-ß Peptide.

Immunoprecipitation (IP) combined with MALDI-TOF mass spectrometry is a powerful instrument for peptide and protein identification in biological samples. In this study, the analytical capabilities of MALDI-TOF/TOF mass spectrometry for relative quantitation of isoAsp7 in Aß(1-42) and Aß(1-16) were investigated. The possibility of quantitative determination of isoAsp7 in Aß(1-42) with the detection limit as low as 2 pmol has been demonstrated. The same approach was applied for a shorter peptide Aß(1-16) and resulted in enhanced accuracy (± 3.2%), and lower detection limit (50 fmol). Pilot experiments with artificial cerebrospinal fluid and mouse brain tissue were performed and showed that the proposed IP-MALDI-TOF/TOF approach could be applied for measuring isoAß content in biological fluids and tissues. Additionally, it was shown that 6E10 anti-amyloid antibodies might affect the accuracy of the amyloid-ß quantitation in the presence of the isomerized peptide.