Real-time glioblastoma tumor microenvironment assessment by SpiderMass for improved patient management.

Glioblastoma is a highly heterogeneous and infiltrative form of brain cancer associated with a poor outcome and limited therapeutic effectiveness. The extent of the surgery is related to survival. Reaching an accurate diagnosis and prognosis assessment by the time of the initial surgery is therefore paramount in the management of glioblastoma. To this end, we are studying the performance of SpiderMass, an ambient ionization mass spectrometry technology that can be used in vivo without invasiveness, coupled to our recently established artificial intelligence pipeline. We demonstrate that we can both stratify isocitrate dehydrogenase (IDH)-wild-type glioblastoma patients into molecular sub-groups and achieve an accurate diagnosis with over 90% accuracy after cross-validation. Interestingly, the developed method offers the same accuracy for prognosis. In addition, we are testing the potential of an immunoscoring strategy based on SpiderMass fingerprints, showing the association between prognosis and immune cell infiltration, to predict patient outcome.

Evaluation of antibody-based single cell type imaging techniques coupled to multiplexed imaging of N-glycans and collagen peptides by matrix-assisted laser desorption/ionization mass spectrometry imaging.

The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with single cell spatial omics methods allows for a comprehensive investigation of single cell spatial information and matrisomal N-glycan and extracellular matrix protein imaging. Here, the performance of the antibody-directed single cell workflows coupled with MALDI-MSI are evaluated. Miralys™ photocleavable mass-tagged antibody probes (MALDI-IHC, AmberGen, Inc.), GeoMx DSP® (NanoString, Inc.), and Imaging Mass Cytometry (IMC, Standard BioTools Inc.) were used in series with MALDI-MSI of N-glycans and extracellular matrix peptides on formalin-fixed paraffin-embedded tissues. Single cell omics protocols were performed before and after MALDI-MSI. The data suggests that for each modality combination, there is an optimal order for performing both techniques on the same tissue section. An overall conclusion is that MALDI-MSI studies may be completed on the same tissue section as used for antibody-directed single cell modalities. This work increases access to combined cellular and extracellular information within the tissue microenvironment to enhance research on the pathological origins of disease.

MALDI HiPLEX-IHC: multiomic and multimodal imaging of targeted intact proteins in tissues.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is one of the most widely used methods for imaging the spatial distribution of unlabeled small molecules such as metabolites, lipids and drugs in tissues. Recent progress has enabled many improvements including the ability to achieve single cell spatial resolution, 3D-tissue image reconstruction, and the precise identification of different isomeric and isobaric molecules. However, MALDI-MSI of high molecular weight intact proteins in biospecimens has thus far been difficult to achieve. Conventional methods normally require in situ proteolysis and peptide mass fingerprinting, have low spatial resolution, and typically detect only the most highly abundant proteins in an untargeted manner. In addition, MSI-based multiomic and multimodal workflows are needed which can image both small molecules and intact proteins from the same tissue. Such a capability can provide a more comprehensive understanding of the vast complexity of biological systems at the organ, tissue, and cellular levels of both normal and pathological function. A recently introduced top-down spatial imaging approach known as MALDI HiPLEX-IHC (MALDI-IHC for short) provides a basis for achieving this high-information content imaging of tissues and even individual cells. Based on novel photocleavable mass-tags conjugated to antibody probes, high-plex, multimodal and multiomic MALDI-based workflows have been developed to image both small molecules and intact proteins on the same tissue sample. Dual-labeled antibody probes enable multimodal mass spectrometry and fluorescent imaging of targeted intact proteins. A similar approach using the same photocleavable mass-tags can be applied to lectin and other probes. We detail here several examples of MALDI-IHC workflows designed to enable high-plex, multiomic and multimodal imaging of tissues at a spatial resolution as low as 5 µm. This approach is compared to other existing high-plex methods such as imaging mass cytometry, MIBI-TOF, GeoMx and CODEX. Finally, future applications of MALDI-IHC are discussed.

MALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined.

Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDI-IHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDI-IHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-αSM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.

Highly Multiplexed Immunohistochemical MALDI-MS Imaging of Biomarkers in Tissues.

Immunohistochemistry (IHC) combined with fluorescence microscopy provides an important and widely used tool for researchers and pathologists to image multiple biomarkers in tissue specimens. However, multiplex IHC using standard fluorescence microscopy is generally limited to 3-5 different biomarkers, with hyperspectral or multispectral methods limited to 8. We report the development of a new technology based on novel photocleavable mass-tags (PC-MTs) for facile antibody labeling, which enables highly multiplexed IHC based on MALDI mass spectrometric imaging (MALDI-IHC). This approach significantly exceeds the multiplexity of both fluorescence- and previous cleavable mass-tag-based methods. Up to 12-plex MALDI-IHC was demonstrated on mouse brain, human tonsil, and breast cancer tissues specimens, reflecting the known molecular composition, anatomy, and pathology of the targeted biomarkers. Novel dual-labeled fluorescent PC-MT antibodies and label-free small-molecule mass spectrometric imaging greatly extend the capability of this new approach. MALDI-IHC shows promise for use in the fields of tissue pathology, tissue diagnostics, therapeutics, and precision medicine.

Large Scale Nanoparticle Screening for Small Molecule Analysis in Laser Desorption Ionization Mass Spectrometry.

Nanoparticles (NPs) have been suggested as efficient matrixes for small molecule profiling and imaging by laser-desorption ionization mass spectrometry (LDI-MS), but so far there has been no systematic study comparing different NPs in the analysis of various classes of small molecules. Here, we present a large scale screening of 13 NPs for the analysis of two dozen small metabolite molecules. Many NPs showed much higher LDI efficiency than organic matrixes in positive mode and some NPs showed comparable efficiencies for selected analytes in negative mode. Our results suggest that a thermally driven desorption process is a key factor for metal oxide NPs, but chemical interactions are also very important, especially for other NPs. The screening results provide a useful guideline for the selection of NPs in the LDI-MS analysis of small molecules.

Investigation of the Chemical Interface in the Soybean-Aphid and Rice-Bacteria Interactions Using MALDI-Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is an emerging technology for high-resolution plant biology. It has been utilized to study plant-pest interactions, but limited to the surface interfaces. Here we expand the technology to explore the chemical interactions occurring inside the plant tissues. Two sample preparation methods, imprinting and fracturing, were developed and applied, for the first time, to visualize internal metabolites of leaves in matrix-assisted laser desorption ionization (MALDI)-MSI. This is also the first time nanoparticle-based ionization was implemented to ionize diterpenoid phytochemicals that were difficult to analyze with traditional organic matrices. The interactions between rice-bacterium and soybean-aphid were investigated as two model systems to demonstrate the capability of high-resolution MSI based on MALDI. Localized molecular information on various plant- or pest-derived chemicals provided valuable insight for the molecular processes occurring during the plant-pest interactions. Specifically, salicylic acid and isoflavone based resistance was visualized in the soybean-aphid system and antibiotic diterpenoids in rice-bacterium interactions.

Multiplex MALDI-MS imaging of plant metabolites using a hybrid MS system.

Plant tissues present intriguing systems for study by mass spectrometry imaging, as they exhibit a complex metabolism and a high degree of spatial localization. This chapter presents a methodology for preparation of plant tissue sections for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) analysis and the use of a hybrid mass spectrometer for "multiplex" imaging. The multiplex method described here provides a wide range of analytical information, including high-resolution, accurate mass imaging and tandem MS scans for structural information, all within a single experiment. While this procedure was developed for plant tissues, it can be readily adapted for analysis of other sample types.

Inhibition of the Fe(III)-catalyzed dopamine oxidation by ATP and its relevance to oxidative stress in Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity.

An interface for sensitive analysis of monoamine neurotransmitters by ion-pair chromatography-electrospray ionization-mass spectrometry with continuous online elimination of ion-pair reagents.

A challenge in coupling ion-pair chromatography (IPC) online with electrospray ionization-mass spectrometry (ESI-MS) is that the nonvolatile ion-pair reagent (e.g., alkyl sulfate for amines or tetrabutylammonium for carboxylic acids) in the mobile phase suppresses the ESI-MS signals in the gas phase and their accumulation can clog the MS sampling interface. Consequently, IPC-ESI-MS is conducted either with a volatile ion-pair reagent, which could compromise the analyte separation efficiency, or with a downstream ion-exchange column to rid the ion-pair reagents of the mobile phase. In the latter approach, the limited capacity of ion-exchange columns requires frequent off-line column regeneration, which affects the separation throughput and prohibits long separations from being performed. A dual-valve, dual-ion exchange column interface of IPC-ESI-MS is designed for undisrupted separations and simultaneous column regeneration. Owing to the efficacy in removing the ion-pair reagent, the detection of eluents of monoamine neurotransmitters by an ion trap MS results in the limits of detection of 0.03 µM for dopamine or DA and 0.01 µM for 5-hydroxytryptamine or 5-HT. These values are lower than those obtained with ion trap MS of similar sensitivity when combined with the use of specialized chromatographic columns or sample preconcentration. Excellent reproducibility was attained with repeatedly regenerated ion-exchange columns (RSD = 4-6%) for an extended period of time (RSD < 6% for 6 days). DA and 5-HT in rat straital extracts were analyzed, and our data demonstrate that interferences inherent in the tissues and the ion-pair reagent have been successfully eliminated. This simple interface should be readily amenable to the separation and MS analysis of other types of polar compounds in complex sample media.

Multiplex mass spectrometry imaging for latent fingerprints.

We have previously developed in-parallel data acquisition of orbitrap mass spectrometry (MS) and ion trap MS and/or MS/MS scans for matrix-assisted laser desorption/ionization MS imaging (MSI) to obtain rich chemical information in less data acquisition time. In the present study, we demonstrate a novel application of this multiplex MSI methodology for latent fingerprints. In a single imaging experiment, we could obtain chemical images of various endogenous and exogenous compounds, along with simultaneous MS/MS images of a few selected compounds. This work confirms the usefulness of multiplex MSI to explore chemical markers when the sample specimen is very limited.

Kinetic studies of inhibition of the amyloid beta (1-42) aggregation using a ferrocene-tagged ß-sheet breaker peptide.

The aggregation of amyloidogenic proteins/peptides has been closely linked to the neuropathology of several important neurological disorders. In Alzheimer's disease, amyloid beta (Aß) peptides and their aggregation are believed to be at least partially responsible for the etiology of Alzheimer's disease. The aggregate-inflicted cellular toxicity can be inhibited by short peptides whose sequences are homologous to segments of the Aß(1-42) peptide responsible for ß-sheet stacking (referred to as the ß-sheet breaker peptides). Here, a water-soluble ferrocene (Fc)-tagged ß-sheet breaker peptide, Fc-KLVFFK(6), was used as an electrochemical probe for kinetic studies of the inhibition of the Aß(1-42) fibrillation process and for determination of the optimal concentration of ß-sheet breaker peptide for efficient inhibition. Our results demonstrate that Fc-KLVFFK(6) interacts with the Aß aggregates instantaneously in solution, and a sub-stoichiometric amount of Fc-KLVFFK(6) is sufficient to inhibit the formation of the Aß oligomers and fibrils and to reduce the toxicity of Aß(1-42). The interaction between Fc-KLVFFK(6) and Aß(1-42) follows a pseudo-first-order reaction, with a rate constant of 1.89 ± 0.05 × 10(-4) s(-1). Tagging ß-sheet breaker peptides with a redox label facilitates design, screening, and rational use of peptidic inhibitors for impeding/altering Aß aggregation.

Separation of intermediates of iron-catalyzed dopamine oxidation reactions using reversed-phase ion-pairing chromatography coupled in tandem with UV-visible and ESI-MS detections.

Reversed-phase ion-pairing chromatography (RP-IPC) is coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) through an interface comprising a four-way switch valve and an anion exchange column. Regeneration of the anion exchange column can be accomplished on-line by switching the four-way switch valve to interconnect the column to a regeneration solution. Positioning the anion exchange column between the RP-IPC and ESI-MS instruments allows the ion-pairing reagent (IPR) sodium octane sulfonate to be removed. The IPC-ESI-MS method enabled us to separate and detect four intermediates of the Fe(III)-catalyzed dopamine oxidation. In particular, 6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe(III)-catalyzed dopamine oxidation becomes possible.

Impaired reward learning and intact motivation after serotonin depletion in rats.

Aside from the well-known influence of serotonin (5-hydroxytryptamine, 5-HT) on emotional regulation, more recent investigations have revealed the importance of this monoamine in modulating cognition. Parachlorophenylalanine (PCPA) depletes 5-HT by inhibiting tryptophan hydroxylase, the enzyme required for 5-HT synthesis and, if administered at sufficiently high doses, can result in a depletion of at least 90% of the brain's 5-HT levels. The present study assessed the long-lasting effects of widespread 5-HT depletions on two tasks of cognitive flexibility in Long Evans rats: effort discounting and reversal learning. We assessed performance on these tasks after administration of either 250 or 500 mg/kg PCPA or saline (SAL) on two consecutive days. Consistent with a previous report investigating the role of 5-HT on effort discounting, pretreatment with either dose of PCPA resulted in normal effortful choice: All rats continued to climb tall barriers to obtain large rewards and were not work-averse. Additionally, rats receiving the lower dose of PCPA displayed normal reversal learning. However, despite intact motivation to work for food rewards, rats receiving the largest dose of PCPA were unexpectedly impaired relative to SAL rats on the pretraining stages leading up to reversal learning, ultimately failing to approach and respond to the stimuli associated with reward. High performance liquid chromatography (HPLC) with electrochemical detection confirmed 5-HT, and not dopamine, levels in the ventromedial frontal cortex were correlated with this measure of associative reward learning.

Reaction rates and mechanism of the ascorbic acid oxidation by molecular oxygen facilitated by Cu(II)-containing amyloid-beta complexes and aggregates.

A forefront of the research on Alzheimer's disease (AD) is the interaction of amyloid beta (Abeta) peptides with redox metal ions (e.g., Cu(II), Fe(III), and Fe(II)) and the biological relevance of the Abeta-metal complexes to neuronal cell loss and homeostasis of essential metals and other cellular species. This work is concerned with the kinetic and mechanistic studies of the ascorbic acid oxidation reaction by molecular oxygen that is facilitated by Cu(II) complexes with Abeta(1-16), Abeta(1-42), and aggregates of Abeta(1-42). The reaction rate was found to linearly increase with the concentrations of Abeta-Cu(II) and dissolved oxygen and be invariant with high ascorbic acid concentrations. The rate constants were measured to be 117.2 +/- 15.4 and 15.8 +/- 2.8 M(-1) s(-1) at low (<100 muM) and high AA concentrations, respectively. Unlike free Cu(II), in the presence of AA, Abeta-Cu(II) complexes facilitate the reduction of oxygen by producing H(2)O(2) as a major product. Such a conclusion is drawn on the basis that the reaction stoichiometry between AA and O(2) is 1:1 when the Abeta concentration is kept at a much greater value than that of Cu(II). A mechanism is proposed for the AA oxidation in which the oxidation states of the copper center in the Abeta complex alternates between 2+ and 1+. The catalytic activity of Cu(II) toward O(2) reduction was found to decrease in the order of free Cu(II) > Abeta(1-16)-Cu(II) > Abeta(1-42)-Cu(II) > Cu(II) complexed by the Abeta oligomer/fibril mixture > Cu(II) in Abeta fibrils. The finding that Cu(II) in oligomeric and fibrous Abeta aggregates possesses considerable activity toward H(2)O(2) generation is particularly significant, since in senile plaques of AD patients the coexisting copper and Abeta aggregates have been suggested to inflict oxidative stress through the production of reactive oxygen species (ROS). Although Cu(II) bound to oligomeric and fibrous Abeta aggregates is less effective than free Cu(II) and the monomeric Abeta-Cu(II) complex in producing ROS, in vivo the Cu(II)-containing Abeta oligomers and fibrils might be more biologically relevant given their stronger association with cell membranes and the closer proximity of ROS to cell membranes.