Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

Oxazolone mediated peptide chain extension and homochirality in aqueous microdroplets.

Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here, we show that i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; ii) addition of water to oxazolone forms the dipeptide; and iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone product, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate pure homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.

High-throughput analysis of tissue microarrays using automated desorption electrospray ionization mass spectrometry.

Tissue microarrays (TMAs) are commonly used for the rapid analysis of large numbers of tissue samples, often in morphological assessments but increasingly in spectroscopic analysis, where specific molecular markers are targeted via immunostaining. Here we report the use of an automated high-throughput system based on desorption electrospray ionization (DESI) mass spectrometry (MS) for the rapid generation and online analysis of high-density (6144 samples/array) TMAs, at rates better than 1 sample/second. Direct open-air analysis of tissue samples (hundreds of nanograms) not subjected to prior preparation, plus the ability to provide molecular characterization by tandem mass spectrometry (MS/MS), make this experiment versatile and applicable to both targeted and untargeted analysis in a label-free manner. These capabilities are demonstrated in a proof-of-concept study of frozen brain tissue biopsies where we showcase (i) a targeted MS/MS application aimed at identification of isocitrate dehydrogenase mutation in glioma samples and (ii) an untargeted MS tissue type classification using lipid profiles and correlation with tumor cell percentage estimates from histopathology. The small sample sizes and large sample numbers accessible with this methodology make for a powerful analytical system that facilitates the identification of molecular markers for later use in intraoperative applications to guide precision surgeries and ultimately improve patient outcomes.

Aqueous microdroplets enable abiotic synthesis and chain extension of unique peptide isomers from free amino acids.

Amide bond formation, the essential condensation reaction underlying peptide synthesis, is hindered in aqueous systems by the thermodynamic constraints associated with dehydration. This represents a key difficulty for the widely held view that prebiotic chemical evolution leading to the formation of the first biomolecules occurred in an oceanic environment. Recent evidence for the acceleration of chemical reactions at droplet interfaces led us to explore aqueous amino acid droplet chemistry. We report the formation of dipeptide isomer ions from free glycine or L-alanine at the air-water interface of aqueous microdroplets emanating from a single spray source (with or without applied potential) during their flight toward the inlet of a mass spectrometer. The proposed isomeric dipeptide ion is an oxazolidinone that takes fully covalent and ion-neutral complex forms. This structure is consistent with observed fragmentation patterns and its conversion to authentic dipeptide ions upon gentle collisions and for its formation from authentic dipeptides at ultra-low concentrations. It also rationalizes the results of droplet fusion experiments that show that the dipeptide isomer facilitates additional amide bond formation events, yielding authentic tri- through hexapeptides. We propose that the interface of aqueous microdroplets serves as a drying surface that shifts the equilibrium between free amino acids in favor of dehydration via stabilization of the dipeptide isomers. These findings offer a possible solution to the water paradox of biopolymer synthesis in prebiotic chemistry.

Desorption Electrospray Ionization Mass Spectrometry Assay for Label-Free Characterization of SULT2B1b Enzyme Kinetics.

The sulfotransferase (SULT) 2B1b, which catalyzes the sulfonation of 3ß-hydroxysteroids, has been identified as a potential target for prostate cancer treatment. However, a major limitation for SULT2B1b-targeted drug discovery is the lack of robust assays compatible with high-throughput screening and inconsistency in reported kinetic data. For this reason, we developed a novel label-free assay based on high-throughput (>1 Hz) desorption electrospray ionization mass spectrometry (DESI-MS) for the direct quantitation of the sulfoconjugated product (CV<10 %; <1 ng analyte). The performance of this DESI-based assay was compared against a new fluorometric coupled-enzyme method that we also developed. Both methodologies provided consistent kinetic data for the reaction of SULT2B1b with its major substrates, indicating the affinity trend pregnenolone>DHEA>cholesterol, for both the phospho-mimetic and wild-type SULT2B1b forms. The novel DESI-MS assay developed here is likely generalizable to other drug discovery efforts and is particularly promising for identification of SULT2B1b inhibitors with potential as prostate cancer therapeutics.

Reaction Acceleration Promoted by Partial Solvation at the Gas/Solution Interface.

The kinetics of organic reactions of different types in microvolumes (droplets, thin films, and sealed tubes) show effects of gas/solution interfacial area, reaction molecularity and solvent polarity. Partial solvation at the gas/solution interface is a major contributor to the 104 -fold reaction acceleration seen in bimolecular but not unimolecular reactions in microdroplets. Reaction acceleration can be used to manipulate selectivity by solvent choice.

Metabolic profiles of human brain parenchyma and glioma for rapid tissue diagnosis by targeted desorption electrospray ionization mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) is well suited for intraoperative tissue analysis since it requires little sample preparation and offers rapid and sensitive molecular diagnostics. Currently, intraoperative assessment of the tumor cell percentage of glioma biopsies can be made by measuring a single metabolite, N-acetylaspartate (NAA). The inclusion of additional biomarkers will likely improve the accuracy when distinguishing brain parenchyma from glioma by DESI-MS. To explore this possibility, mass spectra were recorded for extracts from 32 unmodified human brain samples with known pathology. Statistical analysis of data obtained from full-scan and multiple reaction monitoring (MRM) profiles identified discriminatory metabolites, namely gamma-aminobutyric acid (GABA), creatine, glutamic acid, carnitine, and hexane-1,2,3,4,5,6-hexol (abbreviated as hexol), as well as the established biomarker NAA. Brain parenchyma was readily differentiated from glioma based on these metabolites as measured both in full-scan mass spectra and by the intensities of their characteristic MRM transitions. New DESI-MS methods (5 min acquisition using full scans and MS/MS), developed to measure ion abundance ratios among these metabolites, were tested using smears of 29 brain samples. Ion abundance ratios based on signals for GABA, creatine, carnitine, and hexol all had sensitivities > 90%, specificities > 80%, and accuracies > 85%. Prospectively, the implementation of diagnostic ion abundance ratios should strengthen the discriminatory power of individual biomarkers and enhance method robustness against signal fluctuations, resulting in an improved DESI-MS method of glioma diagnosis.

Intraoperative Mass Spectrometry Platform for IDH Mutation Status Prediction, Glioma Diagnosis, and Estimation of Tumor Cell Infiltration.

BACKGROUND: Surgical tumor resection is the primary treatment option for diffuse glioma, the most common malignant brain cancer. The intraoperative diagnosis of gliomas from tumor core samples can be improved by use of molecular diagnostics. Further, residual tumor at surgical margins is a primary cause of tumor recurrence and malignant progression. This study evaluates a desorption electrospray ionization mass spectrometry (DESI-MS) system for intraoperative isocitrate dehydrogenase (IDH) mutation assessment, estimation of tumor cell infiltration as tumor cell percentage (TCP), and disease status. This information could be used to enhance the extent of safe resection and so potentially improve patient outcomes. METHODS: A mobile DESI-MS instrument was modified and used in neurosurgical operating rooms (ORs) on a cohort of 49 human subjects undergoing craniotomy with tumor resection for suspected diffuse glioma. Small tissue biopsies (ntotal = 203) from the tumor core and surgical margins were analyzed by DESI-MS in the OR and classified using univariate and multivariate statistical methods. RESULTS: Assessment of IDH mutation status using DESI-MS/MS to measure 2-hydroxyglutarate (2-HG) ion intensities from tumor cores yielded a sensitivity, specificity, and overall diagnostic accuracy of 89, 100, and 94%, respectively (ncore = 71). Assessment of TCP (categorized as low or high) in tumor margin and core biopsies using N-acetyl-aspartic acid (NAA) intensity provided a sensitivity, specificity, and accuracy of 91, 76, and 83%, respectively (ntotal = 203). TCP assessment using lipid profile deconvolution provided sensitivity, specificity, and accuracy of 76, 85, and 81%, respectively (ntotal = 203). Combining the experimental data and using PCA-LDA predictions of disease status, the sensitivity, specificity, and accuracy in predicting disease status are 63%, 83%, and 74%, respectively (ntotal = 203). CONCLUSIONS: The DESI-MS system allowed for identification of IDH mutation status, glioma diagnosis, and estimation of tumor cell infiltration intraoperatively in a large human glioma cohort. This methodology should be further refined for clinical diagnostic applications.

Intraoperative detection of isocitrate dehydrogenase mutations in human gliomas using a miniature mass spectrometer.

Knowledge of the isocitrate dehydrogenase (IDH) mutation status of glioma patients could provide insights for decision-making during brain surgery. However, pathology is not able to provide such information intraoperatively. Here we describe the first application of a miniature mass spectrometer (MS) to the determination of IDH mutation status in gliomas intraoperatively. The instrumentation was modified to be compatible with use in the operating room. Tandem MS was performed on the oncometabolite, 2-hydroxyglutarate, and a reference metabolite, glutamate, which is not involved in the IDH mutation. Ratios of fragment ion intensities were measured to calculate an IDH mutation score, which was used to differentiate IDH mutant and wild-type tissues. The results of analyzing 25 biopsies from 13 patients indicate that reliable determination of IDH mutation status was achieved (p = 0.0001, using the Kruskal-Wallis non-parametric test). With its small footprint and low power consumption and noise level, this application of miniature mass spectrometers represents a simple and cost-effective platform for an important intraoperative measurement. Graphical abstract.

High-Throughput Bioassays using "Dip-and-Go" Multiplexed Electrospray Mass Spectrometry.

A multiplexed system based on inductive nanoelectrospray mass spectrometry (nESI-MS) has been developed for high-throughput screening (HTS) bioassays. This system combines inductive nESI and field amplification micro-electrophoresis to achieve a "dip-and-go" sample loading and purification strategy that enables nESI-MS based HTS assays in 96-well microtiter plates. The combination of inductive nESI and micro-electrophoresis makes it possible to perform efficient in situ separations and clean-up of biological samples. The sensitivity of the system is such that quantitative analysis of peptides from 1-10 000 nm can be performed in a biological matrix. A prototype of the automation system has been developed to handle 12 samples (one row of a microtiter plate) at a time. The sample loading and electrophoretic clean-up of biosamples can be done in parallel within 20 s followed by MS analysis at a rate of 1.3 to 3.5 s per sample. The system was used successfully for the quantitative analysis of BACE1-catalyzed peptide hydrolysis, a prototypical HTS assay of relevance to drug discovery. IC50 values for this system were in agreement with LC-MS but recorded in times more than an order of magnitude shorter.

Metabolites and Lipids Associated with Fetal Swine Anatomy via Desorption Electrospray Ionization - Mass Spectrometry Imaging.

Chemical imaging by mass spectrometry (MS) has been largely used to study diseases in animals and humans, especially cancer; however, this technology has been minimally explored to study the complex chemical changes associated with fetal development. In this work, we report the histologically-compatible chemical imaging of small molecules by desorption electrospray ionization (DESI) - MS of a complete swine fetus at 50 days of gestation. Tissue morphology was unperturbed by morphologically-friendly DESI-MS analysis while allowing detection of a wide range of small molecules. We observed organ-dependent localization of lipids, e.g. a large diversity of phosphatidylserine lipids in brain compared to other organs, as well as metabolites such as N-acetyl-aspartic acid in the developing nervous system and N-acetyl-L-glutamine in the heart. Some lipids abundant in the lungs, such as PC(32:0) and PS(40:6), were  similar to surfactant composition reported previously. Sulfatides were highly concentrated in the fetus liver, while hexoses were barely detected at this organ but were abundant in lung and heart. The chemical information on small molecules recorded via DESI-MS imaging coupled with traditional anatomical evaluation is a powerful source of bioanalytical information which reveals the chemical changes associated with embryonic and fetal development that, when disturbed, causes congenital diseases such as spina bifida and cleft palate.

Selective Gas-Phase Mass Tagging via Ion/Molecule Reactions Combined with Single Analyzer Neutral Loss Scans to Probe Pharmaceutical Mixtures.

We have demonstrated the use of a simple single ion trap mass spectrometer to identify classes of compounds as well as individual components in complex mixtures. First, a neutral reagent was used to mass tag oxygen-containing analytes using a gas-phase ion/molecule reaction. Then, a neutral loss scan was used to indicate the carboxylic acids. The lack of unit mass selectivity in the neutral loss scan required subsequent product ion scans to confirm the presence and identity of the individual carboxylic acids. The neutral loss scan technique reduced the number of data-dependent MS/MS scans required to confirm identification of signals as protonated carboxylic acids. The method was demonstrated on neat mixtures of standard carboxylic acids as well as on solutions of relevant pharmaceutical tablets and may be generalizable to other ion/molecule reactions.

Rapid determination of isocitrate dehydrogenase mutation status of human gliomas by extraction nanoelectrospray using a miniature mass spectrometer.

Isocitrate dehydrogenase (IDH) I and II mutations in gliomas cause an abnormal accumulation of 2-hydroxyglutarate (2-HG) in these tumor cells. These mutations have potential prognostic value in that knowledge of the mutation status can lead to improved surgical resection. Information on mutation status obtained by immunohistochemistry or genomic analysis is not available during surgery. We report a rapid extraction nanoelectrospray ionization (nESI) method of determining 2-HG. This should allow the determination of IDH mutation status to be performed intraoperatively, within minutes, using a miniature mass spectrometer. This study demonstrates that the combination of tandem mass spectrometry with low-resolution mass spectrometry allows this analysis to be performed with confidence. Graphical Abstract.

Intraoperative assessment of isocitrate dehydrogenase mutation status in human gliomas using desorption electrospray ionization-mass spectrometry.

OBJECTIVE: The authors describe a rapid intraoperative ambient ionization mass spectrometry (MS) method for determining isocitrate dehydrogenase (IDH) mutation status from glioma tissue biopsies. This method offers new glioma management options and may impact extent of resection goals. Assessment of the IDH mutation is key for accurate glioma diagnosis, particularly for differentiating diffuse glioma from other neoplastic and reactive inflammatory conditions, a challenge for the standard intraoperative diagnostic consultation that relies solely on morphology. METHODS: Banked glioma specimens (n = 37) were analyzed by desorption electrospray ionization-MS (DESI-MS) to develop a diagnostic method to detect the known altered oncometabolite in IDH-mutant gliomas, 2-hydroxyglutarate (2HG). The method was used intraoperatively to analyze tissue smears obtained from glioma patients undergoing resection and to rapidly diagnose IDH mutation status (< 5 minutes). Fifty-one tumor core biopsies from 25 patients (14 wild type [WT] and 11 mutant) were examined and data were analyzed using analysis of variance and receiver operating characteristic curve analysis. RESULTS: The optimized DESI-MS method discriminated between IDH-WT and IDH-mutant gliomas, with an average sensitivity and specificity of 100%. The average normalized DESI-MS 2HG signal was an order of magnitude higher in IDH-mutant glioma than in IDH-WT glioma. The DESI 2HG signal intensities correlated with independently measured 2HG concentrations (R2 = 0.98). In 1 case, an IDH1 R132H-mutant glioma was misdiagnosed as a demyelinating condition by frozen section histology during the intraoperative consultation, and no resection was performed pending the final pathology report. A second craniotomy and tumor resection was performed after the final pathology provided a diagnosis most consistent with an IDH-mutant glioblastoma. During the second craniotomy, high levels of 2HG in the tumor core biopsies were detected. CONCLUSIONS: This study demonstrates the capability to differentiate rapidly between IDH-mutant gliomas and IDH-WT conditions by DESI-MS during tumor resection. DESI-MS analysis of tissue smears is simple and can be easily integrated into the standard intraoperative pathology consultation. This approach may aid in solving differential diagnosis problems associated with low-grade gliomas and could influence intraoperative decisions regarding extent of resection, ultimately improving patient outcome. Research is ongoing to expand the patient cohort, systematically validate the DESI-MS method, and investigate the relationships between 2HG and tumor heterogeneity.

Fatty Acid Patterns Detected By Ambient Ionization Mass Spectrometry in Canine Invasive Urothelial Carcinoma From Dogs of Different Breeds.

BACKGROUND: In early work ambient ionization mass spectrometry (MS) revealed lipid patterns distinguishing muscle invasive bladder cancer (invasive urothelial carcinoma, InvUC) from normal urothelium. A new ambient ionization MS approach, touch spray MS (TS-MS) can rapidly generate mass spectra in real time, potentially in a point-of-care setting. A tissue sample removed from a patient is touched by a probe, and mass spectra generated within seconds. OBJECTIVE: To validate TS-MS methods using specimens from naturally-occurring InvUC in dogs where the cancer closely mimics the human condition, and to demonstrate proof-of-concept that TS-MS can elucidate lipid patterns distinguishing InvUC from normal urothelium. METHODS: Samples of normal urothelium and InvUC from dogs of several breeds were analyzed by TS-MS with correlative histopathology across each sample. Results were compared to those obtained with desorption electrospray ionization mass spectrometry (DESI-MS), a more traditional method. Data were analyzed by Principal Component Analysis and Linear Discriminant Analysis. RESULTS: Lipid patterns identified by TS-MS, as well as by DESI-MS, differed between InvUC and normal urothelium with m/z 281.5 (oleic acid) and m/z 563.5 (oleic acid dimer) substantially contributing to the differences. Using histologic diagnosis as the gold standard, TS-MS had a global prediction rate of 93%. CONCLUSIONS: TS-MS can be used to identify lipid patterns that differentiate canine InvUC from normal urothelium. Optimization of TS-MS could lead to a point-of-care approach to distinguish cancer from normal in ex vivo tissues in real time, and to define biochemical processes leading to cancer development and progression.

Mass Spectrometry for Synthesis and Analysis.

Mass spectrometry is the science and technology of ions. As such, it is concerned with generating ions, measuring their properties, following their reactions, isolating them, and using them to build and transform materials. Instrumentation is an essential element of these activities, and analytical applications are one driving force. Work from the Aston Laboratories at Purdue University's Department of Chemistry is described here, with an emphasis on accelerated reactions of ions in solution and small-scale synthesis; ion/surface collision processes, including surface-induced dissociation (SID) and ion soft landing; and applications to tissue imaging. Our special interest in chirality and the chemistry behind the origins of life is also featured together with the exciting area of tissue diagnostics.

Feasibility of desorption electrospray ionization mass spectrometry for diagnosis of oral tongue squamous cell carcinoma.

RATIONALE: Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated utility in differentiating tumor from adjacent normal tissue in both urologic and neurosurgical specimens. We sought to evaluate if this technique had similar accuracy in differentiating oral tongue squamous cell carcinoma (SCC) from adjacent normal epithelium due to current issues with late diagnosis of SCC in advanced stages. METHODS: Fresh frozen samples of SCC and adjacent normal tissue were obtained by surgical resection. Resections were analyzed using DESI-MS sometimes by a blinded technologist. Normative spectra were obtained for separate regions containing SCC or adjacent normal epithelium. Principal Component Analysis and Linear Discriminant Analysis (PCA-LDA) of spectra were used to predict SCC versus normal tongue epithelium. Predictions were compared with pathology to assess accuracy in differentiating oral SCC from adjacent normal tissue. RESULTS: Initial PCA score and loading plots showed clear separation of SCC and normal epithelial tissue using DESI-MS. PCA-LDA resulted in accuracy rates of 95% for SCC versus normal and 93% for SCC, adjacent normal and normal. Additional samples were blindly analyzed with PCA-LDA pixel-by-pixel predicted classifications as SCC or normal tongue epithelial tissue and compared against histopathology. The m/z 700-900 prediction model showed a 91% accuracy rate. CONCLUSIONS: DESI-MS accurately differentiated oral SCC from adjacent normal epithelium. Classification of all typical tissue types and pixel predictions with additional classifications should increase confidence in the validation model.

Analysis of human gliomas by swab touch spray-mass spectrometry: applications to intraoperative assessment of surgical margins and presence of oncometabolites.

Touch spray mass spectrometry using medical swabs is an ambient ionization technique (ionization of unprocessed sample in the open air) that has potential intraoperative application in quickly identifying the disease state of tissue and in better characterizing the resection margin. To explore this potential, we studied 29 human brain tumor specimens and obtained evidence that this technique can provide diagnostic molecular information that is relevant to brain cancer. Touch spray using medical swabs involves the physical sampling of tissue using a medical swab on a spatial scale of a few mm2 with subsequent ionization occurring directly from the swab tip upon addition of solvent and application of a high voltage. Using a tertiary mixture of acetonitrile, N,N-dimethylformamide, and ethanol, membrane-derived phospholipids and oncometabolites are extracted from the tissue, incorporated into the sprayed microdroplets, vacuumed into the mass spectrometer, and characterized in the resulting mass spectra. The tumor cell load was assessed from the complex phospholipid pattern in the mass spectra and also separately by measurement of N-acetylaspartate. Mutation status of the isocitrate dehydrogenase gene was determined via detection of the oncometabolite 2-hydroxyglutarate. The lack of sample pretreatment makes touch spray mass spectrometry using medical swabs a feasible intraoperative strategy for rapid surgical assessment.