Laser Desorption Ionization Mass Research Articles

Mass spectrometry study of ascorbyl palmitate as an agent for nanosomes formation

Background. Study of properties and intermolecular interactions of biologically active compounds which can be used for the purposes of transmembrane drugs delivery is a topical task of modern molecular biophysics. Ascorbyl Palmitate (AP) as a fat-soluble form of vitamin C has recently attracted attention as a promising agent for formation of nanosomes for the “fat insoluble” drug molecules transfer through membranes. However, AP is not sufficiently characterized by up-to-date soft ionization mass spectrometric techniques. Objectives. The aim of the present work is to characterize AP and its intermolecular interactions by a number of mass spectrometric techniques: Electrospray Ionization (ESI), Laser Desorption/Ionization (LDI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). The comparison of these techniques applicability to the study of AP intermolecular interactions as a drug delivery assisting agent is scheduled. Methods. ESI mass spectra are obtained with triple quadrupole Micromass Quattro mass spectrometer. LDI and MALDI experiments are performed by Autoflex II mass spectrometer. Results. In the ESI experiments in the positive ion mode abundant peaks of protonated and cationized AP molecules as well as the peaks of AP clusters nAP•H+ and nAP•Na+ (n=2÷4) are revealed in the mass spectra. This result testifies to the formation of stable noncovalent complexes of the AP molecules in the polar media and confirms the AP ability of formation nanosomes for drug delivery. Analysis of LDI and MALDI mass spectra of AP in positive and negative ion modes shows that in the presence of molecular ions of AP, the peaks of AP dimers or larger AP clusters are not recorded. The ESI probing of the model system containing AP and dipalmitoylphosphatidylcholine (DPPC) reveals stable AP•DPPC•H+ complex which models the AP intermolecular interactions with the phospholipid components of biomembranes and/or liposomes under AP functioning as a drug delivery assisting agent. Conclusions. The current study demonstrates the applicability of all tested mass spectrometric techniques for AP identification in solutions and solid phase, while for the purpose of examining of the AP noncovalent complexes formation and study of AP interactions with biomolecules the ESI is defined as the most effective technique.

An insight into spray paints for street art: Chemical characterization of two yellow varnishes by spectroscopic and MS-based spectrometric techniques

In recent decades, modern murals and graffiti have become valuable artistic expressions that deserve attention for their dissemination and conservation. The chemical composition of spray paints with improved technical performance reflects the assortment of materials used in these artworks. Understanding the spray formulations is important in preventing deterioration of street art. In this study, two yellow varnishes were investigated by attenuated total reflectance Fourier-transform infrared (ATR-FTIR) and X-ray photoelectron spectroscopies (XPS), laser desorption ionization (LDI) and matrix-assisted (MA) LDI mass spectrometry (MS). ATR-FTIR allowed us to distinguish the occurrence of acrylic and styrene-acrylic resins as the main binders with some additional components, while XPS shed light on the recognition of some silicone-based additives, which aggregate at the most superficial varnish layers. Several established experimental conditions of MALDI-MS including matrix, salt dopant and extraction solvent, revealed the use of polyethylene glycol as an additive, while direct LDI-MS/MS analysis proved to be very useful in characterizing the yellow (PY74) and orange (PO36) pigments. Some insight into the minor components of the spray paints, such as surfactant, humectant and silicone-based levelling or anti-cratering agents, which were not easily obtained through conventional approaches, was also gained.

Abstract CT565: Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer

Abstract Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Detection of Acetaminophen and Its Glucuronide in Fingerprint by SALDI Mass Spectrometry Using Zeolite and Study of Time-Dependent Changes in Detected Ion Amount

The ingredients of an antipyretic (acetaminophen, AAP) and their metabolites excreted into fingerprint were detected by surface-assisted laser desorption ionization (SALDI) mass spectrometry using zeolite. In the fingerprint taken 4 h after AAP ingestion, not only AAP but also the glucuronic acid conjugate of AAP (GAAP), caffeine (Caf), ethenzamide (Eth), salicylamide (Sala; a metabolite of Eth), and urea were detected. Fingerprints were collected over time to determine how the amounts of AAP and its metabolite changed with time, and the time dependence of the peak intensities of protonated AAP and GAAP was measured. It was found that the increase of [GAAP+H]+ peak started later than that of [AAP+H]+ peak, reflecting the metabolism of AAP. Both AAP and GAAP reached maximum concentrations approximately 3 h after ingestion, and were excreted from the body with a half-life of approximately 3.3 h. In addition, fingerprint preservation was confirmed by optical microscopy, and fingerprint shape was retained even after laser irradiation of the fingerprint. Our method may be used in fingerprint analysis.

Physicochemical properties and cytotoxicity of brown carbon produced under different combustion conditions

Light-absorbing organic particulate matter (PM), or brown carbon (BrC), may constitute an important fraction of combustion PM. Here, we investigate the effect of combustion conditions on the molecular sizes of BrC, their light-absorption properties, and their cytotoxicity. We used toluene in a combustion reactor with highly controlled conditions to produce two different types of BrC under two conditions corresponding to smoldering and near-flaming combustion, with temperatures of 670 °C and 1035 °C, respectively. We performed online measurements of the size distributions and light-absorption properties of the BrC. The BrC produced at 1035 °C was more light absorbing, with an imaginary component of the refractive index at 532 nm (k532) an order of magnitude larger than that of the BrC produced at 670 °C. We also collected samples for offline chemical characterization using laser desorption ionization (LDI) mass spectrometry. The LDI mass spectra showed that the BrC produced at 1035 °C was composed of species with significantly larger molecular sizes than the BrC produced at 670 °C. Using human lung epithelial cells, we conducted in vitro cytotoxicity analysis on the two types of BrC with doses ranging from 3.5 to 136.0 μg of BrC/ml. After 24-h exposure, the viability of the cells was assessed using a WST-8 assay. The cytotoxicity analysis showed that, for both BrC samples, the cells exhibited a clear dose-dependent response with significant BrC cytotoxicity that plateaued at the higher doses. However, while the viability of cells exposed to the BrC produced at 1035 °C reached a minimum of around 65% at the highest dose, the BrC produced at 670 °C proved to be significantly more toxic, with the viability dropping asymptotically to 25%. The results presented here suggest that organic PM of smaller molecular sizes produced under lower temperature, smoldering combustion could be significantly more toxic than that of larger molecular sizes produced under higher temperature, flaming conditions. The use of a single-molecule fuel in a highly controlled combustion setup distinguishes this work from experiments that rely on real-life sources and combustion setups, where different combustion conditions could be occurring simultaneously and clouding the conclusions.

High-resolution imaging mass spectrometry combined with transcriptomic analysis identified a link between fatty acid composition of phosphatidylinositols and the immune checkpoint pathway at the primary tumour site of breast cancer

BackgroundThe fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS).MethodsWe visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles.ResultsNormal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition.ConclusionsMALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.

Synergistic Effect of the Heterostructure of Au Nanoislands on TiO2 Nanowires for Efficient Ionization in Laser Desorption/Ionization Mass Spectrometry.

A combination nanostructured matrix with metal Au nanoislands and semiconductor TiO2 nanowires is presented to enhance both desorption and ionization efficiency in laser desorption/ionization (LDI) mass spectrometry. The heterostructure of Au nanoislands on TiO2 nanowires was fabricated via (1) TiO2 nanowire synthesis through a modified wet-corrosion method and (2) Au nanoisland formation through thermal annealing of a sputtered Au layer on the TiO2 nanowires. Herein, the synergistic effect of this heterostructure for highly efficient ion production was experimentally elucidated in terms of the formation of high temperature on the surface of Au and the creation of a Schottky barrier at the Au-TiO2 interface. Finally, four types of immunosuppressors were analyzed to demonstrate the improved ionization performance of the heterostructure for LDI mass spectrometry.

Metal-organic framework-based affinity materials in proteomics.

Metal-organic frameworks (MOFs) are an eminent addition to materials science research because of their versatile properties due to which their applications are wide spread in proteomics. They are used in various fields due to their characteristics like higher surface area, specific symmetry, ease of modification, and availability of a variety of ligands. As affinity sorbents, they have shown higher selectivity, sensitivity, and reproducibility than conventionally used materials. They are applied for the enrichment of phosphopeptides, glycopeptides, low mass peptides, and as laser desorption/ionization (LDI) matrices for small-molecule analysis. This review captures the insight of applying MOFs in the field of mass spectrometry-based proteomics. The specific features are discussed regarding MOFs as affinity sorbents for the selective capture of biological molecules like phosphopeptides and glycopeptides from complex samples. The potential of MOFs as LDI mass spectrometry (LDI-MS) matrices for small-molecule analysis is also evaluated. MOFs have also been used as enzymatic reactors for the digestion of proteins, prior to MS analysis. MOF-based affinity materials and bioreactors reduce proteome complexity and improve detection sensitivity and coverage. Size-exclusion effects of MOFs help in subtracting the abundant proteins in peptidomics. Several limitations of MOFs are addressed, which include stability under varying pH conditions, the unclear interaction mechanism between the MOFs and targeted analytes, and the non-specific binding that interferes during the analysis because of metal centers and ligands in the MOFs. This will open up MOF-based research to overcome the limitations and improve the performance of MOFs as selective and sensitive materials. Graphical abstract ᅟ.

In English

During last decades сoordination compounds of lanthanides with polychelating β-diketones and their derivatives have attracted increasing interest due to the possibility of using such compounds in biomedical research, as contrast reagents for NMR tomography studies and MOLED technology. That is why the selection of appropriate ligands, which are effective sensitizers of lanthanide luminescence and can form thermodynamically stable complexes, is especially important. Carbacylamidophosphates (CAPh ligands) of general formula (R 1 )C(O)NHP(O)(R 2 ) 2 are structural analogues of β-diketones, which can serve as an antenna providing more efficient energy transfer to the lanthanide ion followed by Ln(III) luminescence. The present work is devoted to the mass-spectrometric studies of the stability of coordination compound of terbium(III) with new CAPh ligand using methods of soft ionization such as matrix activated laser desorption / ionization (MALDI), its modification – LDI (laser desorption ionization). The main preference of the «soft ionization» methods is the possibility of unfragmented ions registration. Terbium coordination compound with new bis-chelating carbacylamidophosphate ligand (Tb(HL) 2 (NO 3 )) (where HL – = tetramethyl[pyridine-2,6-diyldi(iminocarbonyl)]diamidophosphate anion) has been synthesized and studied by IR spectroscopy and X-ray diffraction. It was shown that the deprotonated arm of each ligand is coordinated to the terbiumion in the bidentate manner and the protonated arm in monodentate manner via the oxygen atom of phosphoryl group. The nitrate ion is bidentate coordinated, completing coordination number of terbium to eight. The components of the LDI mass spectrum of the synthesized coordination compound have been identified. The positive region of the mass spectrum of the complex Tb(HL) 2 (NO 3 ) is characterized by the presence of smaller fragments due to the effect of the metal ion on the fragmentation of the ligand molecules and the molecular ion is not observed due to formation of more complex and stable structures based on Tb(III) ions.

Sputter-Coated Metal Screening for Small Molecule Analysis and High-Spatial Resolution Imaging in Laser Desorption Ionization Mass Spectrometry.

Nanoparticles are efficient matrices in laser desorption/ionization (LDI) mass spectrometry (MS), especially for the profiling or imaging of small molecules. Recently, solvent-free physical vapor desorption (PVD), or sputter coating, was adopted as a homogenous method to rapidly apply metal nanoparticles (NPs) in situ to samples prior to LDI MS or MS imaging analysis. However, there has been no systematic study comparing different metal targets for the analysis of a variety of small molecule metabolites. Here, we present a screening and optimization of various sputter-coated metals, including Ag, Au, Cu, Pt, Ni, and Ti, for LDI analysis of small molecules in both positive and negative ion modes. Optimized sputter coating is then applied to high-spatial resolution LDI mass spectrometry imaging (MSI) of maize root and seed cross-sections. Noble metals, Ag, Au, and Pt, are found to be much more efficient than transition metals and organic matrices for most small metabolites. Sputter-coated metals are efficient for neutral lipids, such as triacylglycerols and diacylglycerols, but are very inefficient for most phospholipids. Graphical Abstract ᅟ.

The ongoing evolution of laser desorption/ionization mass spectrometry: Some observations on current trends and future directions

The special feature in this issue of the Journal explores trends, challenges and opportunities in the broad area of laser desorption/ ionization (LDI) mass spectrometry. The authors discuss the potential of novel sample application strategies and modifications to the plate surface to take LDI to another level. Although adaptations of this type have been around almost as long as LDI itself, they have had a checkered past, and until now have not been widely adopted, either by end-users or instrument manufacturers. Muthu and colleagues suggest that this will soon change. A rapidly expanding array of modifications, made possible through advances in surface engineering and nanotechnology, are now poised to change the way we think about and practice LDI mass spectrometry.

Mesoporous organosilica films for laser desorption/ionization mass spectrometry

Mass spectrometry (MS) is a powerful analytical tool that can detect and identify multiple kinds of compounds such as biomolecules with high sensitivity. Recently, various nanoporous solid substrates have been developed for organic-matrix-free laser desorption/ionization (LDI) of analytes. In this study, periodic mesoporous organosilica (PMO) films are demonstrated for the first time to be promising as solid substrates for LDI mass spectrometry (LDI-MS). UV-absorbing PMO thin films having surface open pores with diameters of 20–30 nm are prepared using a triphenylamine derivative as a precursor and an amphiphilic block copolymer as a structure-directing template, followed by nanoscale surface etching. Peptide analytes introduced into the mesopores can be efficiently detected on the order of sub-picomole without the addition of an organic matrix. In addition, the PMO film shows an advantage of superior shot-to-shot reproducibility compared to a conventional matrix-assisted LDI (MALDI) method on a simple and easy way. PMO films with high tunability of framework composition and functionality may pave the way to new solid substrates for LDI-MS.

The effect of antigen retrieval buffers on MALDI mass spectrometry imaging of peptide profiles in skin FFPE tissue

Abstract Objectives: The success of novel in-situ immunohistoproteomics approaches that employ Matrix-assisted laser desorption and ionization (MALDI) mass spectrometry imaging (MSI) heavily depends on good antigen retrieval efficacy. Methods: We have optimized the effect of various antigen retrieval buffers on peptide profiles acquired from FFPE traction alopecia scalp tissue. Heat-induced epitope retrieval (HIER) was performed using Citrate, Tris, and Tris-EDTA buffers under various conditions. MALDI-MSI analysis of the tissue was performed using a high-throughput, state-of-the-art RapiFlex MALDI TOF/TOF tissue-typer equipped with a smart beam laser. Results: Background matrix peaks were found under most conditions except under Tris-EDTA buffer. Trypsin digestion periods longer than overnight and possibly higher concentrations are needed for Citrate and Tris. The Tris-EDTA buffer performed best for MALDI-MSI antigen retrieval at an incubation period of overnight at 60°C. Conclusions: We have identified in this study that antigen retrieval conditions need to be optimised prior to MALDI MSI analysis of different tissue types.

Characterization of the Ag43Cu37Zn20 alloy surface after potentiostatic polarization using LDI-TOF mass spectrometry

AbstractThis work is the first to demonstrate the possibilities of direct analysis of the corrosion film formed on Ag43Cu37Zn20 alloy using laser desorption/ionization (LDI) on a commercial matrix-assisted LDI time of flight mass spectrometry (MS) instrument. In the LDI mass spectra measured from the water solution of the corrosion film, the ion species Zn+, Zn(H2O)+, AgH+, Cu2H+, Cu2O(OH)+, Cu3O(OH)+ (high intensity), and Cu(OH)(H2O)+, ZnCl(OH)(H2O)+, AgCl+, CuCl+ (low intensity) were identified. The LDI mass spectra of the propan-2-ol solution of the corrosion film contains the ions Zn5Cl(OH)3(H2O)11+, Zn5Cl2(OH)8(H2O)6+, and Zn5Cl2(OH)(H2O)14+. Mass spectra were observed at laser energies in the range of 1340–1860 arbitrary units in positive ion mode. Previously, Raman spectra and X-ray diffraction (XRD) analysis of the same anodic film have shown similar results as LDI MS – that the anodic film contains the mixture of Ag, Cu2O, CuCl, Zn5Cl2(OH)8·H2O, and β-Zn(OH)Cl. LDI MS could be a complementary method for XRD and Raman, as demonstrated in this investigation. The LDI MS method greatly reduces the analysis time and amount of the sample compared to traditional methods. For this reason, the LDI MS method can be a useful tool for the fast qualitative screening of corrosion films.

Abstract 4647: Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation

Abstract Introduction: Mutations of the isocitrate dehydrogenase (IDH)-1/2 genes frequently occur in certain malignancies, including gliomas. Mutant IDH-1/2 proteins gain a new ability to produce oncometabolite 2-hydroxiglutarate (2HG), which results in a different clinical impact on gliomas in comparison to those with a wild-type IDH. The status of IDH mutations has been determined by immunohistochemistry (IHC) using the IDH1-R132H antibody or through the DNA sequencing of IDH1-R132C/R132S; however, its practical applications are still under discussion, and the significance of the oncometabolite 2HG evaluation is not clear. In this study, we analyzed the serum and tissue levels of 2HG in clinical gliomas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) system. Method: Glioma samples (n = 78, matched pairs of serum and flash frozen tissue) were obtained between October 2007 to January 2015 at the National Cancer Center Hospital, Japan. High performance liquid chromatography (Nexera X2, Shimadzu) and triple quad mass spectrometry (QTRAP4500, AB SCIEX) were used for LC-MS/MS analysis. The two MALDI-MSI systems, quadrupole ion trap time-to-flight mass spectrometry imaging (iMScope, Shimadzu), and high-resolution atmospheric pressure mass spectrometry imaging (AP-SMALDI, TransMIT; Q-Exactive, Thermo Fisher Scientific) were used for analyzing the 2HG tissue distribution after coating with 9-aminoacridine or 2,5-dihydroxybenzoic acid. The IDH mutation was determined by direct sequencing and IHC. Results: The median 2HG concentration in serum was 21.43 ng/mL (range: 9.58 to 62.51) in this study, and no significant correlation was observed between the IDH mutation and the wild type status (mutation, n = 28, median 20.2 ng/mL, range 9.58 to 62.51 vs. wild type, n = 50, median 22.88 ng/mL, range 11.51 to 40.32; Wilcoxon test p = 0.1597). The median of the 2HG tissue concentration in the entire group was 0.16 ng/μg, and a significant difference was seen between the IDH mutant and wild type groups (mutation, median 4.86 ng/μg, range 0.51 to 49.86 vs. wild type, median 0.09 ng/μg, range 0.02 to 0.45; Wilcoxon test p = <0.0001). In the MALDI-MSI analysis, the average 2HG ion intensities were 381.62/mm2 in the IDH mutation and 8.25/mm2 in the wild-type IDH tissues (Wilcoxon test p = 0.016), which showed a positive correlation with the 2HG tissue concentration by LC-MSMS analyses (Spearman ρ = 0.87, p = 0.0003). The MSI results indicate a possible link between the 2HG tissue distributions and the histology of IDH mutant sections. Conclusion: Tissue 2HG measurement using mass spectrometric analyses may be a useful alternative in determining IDH mutations. Further research may provide a biological perspective and help gain some insights on the clinical significance of 2HG. Citation Format: Mitsuhiro Hayashi, Hiroaki Aikawa, Makoto Ohno, Koichi Ichimura, Yuko Matsushita, Mayu Ohuchi, Mariko Mizui, Akihiko Yoshida, Yoshitaka Narita, Akinobu Hamada. Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2017-4647

Abstract POSTER-TECH-1122: Mass spectrometric imaging of ovarian cancer mouse model tissue: searching for high-grade serous carcinoma biomarkers

Abstract High-grade serous carcinoma (HGSC) is the most common and deadliest ovarian cancer responsible for 90% of all ovarian cancer deaths. Studying the origin and early progression of this deadly cancer is critical in identifying biomarkers for early detection of the disease. Unfortunately, in the case of HGSC, it is virtually impossible to obtain early-stage human HGSC tissue samples because there is no diagnostic method capable of detecting early forms of HGSC. The recent breakthrough of an animal model (Dicer-Pten double-knockout mice) provides an invaluable opportunity to finally investigate the early tumor microenvironment. The model exhibits remarkable similarities to human HGSC, allowing greater insight into the biochemistry associated with nascent neoplastic changes within ovarian and fallopian tube tissues. An important aspect of determining potential biomarkers is to correlate any detected chemical changes with observed histological changes. To visualize the spatial distribution of chemical changes, tissue-based mass spectrometric imaging (MSI) can be employed as an unbiased way of visualizing the changes of proteins, metabolites, or both in cancer tissues. MSI is particularly useful for identifying molecules uniquely elevated or lowered in early tumor tissues, in which cell-type specific changes are expected to occur due to intratumoral microenvironment heterogeneity. In this study, two complementary MSI techniques – matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometry – are applied to thin tissue sections of Dicer-Pten mutant mouse ovaries and fallopian tubes. Several sulfatide lipids d18:1/C16:0, d18:1/C24:1 and d18:1/C24:0 have been implicated as potential biomarkers in human ovarian cancer. These metabolites, among others, are being included in a thorough MSI experiment to visualize molecular changes in multiple cell types in the microenvironment during early tumor progression. In addition to providing information about the early molecular changes taking place, the knowledge gained from this study will also help understand which specific cell types in the fallopian tube are more prone to early tumorigenesis. (Grant support: Marsha Rivkin Ovarian Cancer Challenge Grant) Citation Format: Martin R. L. Paine, Rachel V. Bennett, Jaeyeon Kim, M. Cameron Sullards, Martin M. Matzuk, Facundo M. Fernández. Mass spectrometric imaging of ovarian cancer mouse model tissue: searching for high-grade serous carcinoma biomarkers [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1122.

Visible fluorescence of biological fluids as a renal failure marker: New integrative approach

Solid phases of visible fluorescence substance (VFS) of biological fluids (blood, urine, hemodialysate) which was proposed earlier as a morbidity and mortality marker by renal failure and diabetes were investigated in-depth by the methods of electron and confocal microscopy, optical spectroscopy and matrix assisted laser desorption-ionization (MALDI) mass spectroscopy. It is shown that dry VFS exists predominantly in the form of carbon–oxygen–nitrogen (N ≈ 8.7 wt .%) nanoparticles (NPs) (5 ≤ d ≤ 100 nm ). For the first time the existence of the threshold energy Eg ≈ 2.15 eV for excitation of VFS was observed experimentally and confirmed by semi-empirical calculations of the bathochromic shift. A good accordance with the earlier autonomous theoretical calculations was achieved. Thus, the long wavelength limit (575 nm) of the spectral range where the VFS can be used as a fluorescent marker was reliably determined. A pilot MALDI comparative study of graphene oxide (GO) and urine VFS was carried out. Six kinds of nitrogen-free particles (412 ≤ M ≤ 456 Da ) were observed in each substance and possible computer models of those have been composed. It is established that along with nitrogen-containing advanced glycation end products (AGEs) also nitrogen-free carbon–oxygen–hydrogen particles (probably toxic) with the composition and structure related to GO can exist in biofluids. Both types of particles should be taken into account in search for the reasons of high mortality among end stage renal disease patients.

Direct analysis of lipids from blood using matrix-assisted laser desorption ionization (MALDI) mass spectrometry

Introduction: Lipids are essential biological pathways involved in metabolism, signalling and energy storing. However, abnormal lipid levels, such as cholesterol, play a crucial role in heart diseases and type 2 diabetes mellitus. Knowing cholesterol levels is therefore essential in monitoring the metabolic functions of the individual and preventing chronic and lethal diseases. Objectives: To evaluate the potential of matrixassisted laser desorption ionization (MALDI) mass spectrometry as a fast and reliable method for the direct screening of patients. Materials and methods: An aliquot consisting of 15– 20μl of human blood was collected from the finger. Lipids were extracted from blood either by direct extraction or after a preliminary centrifugation and plasma separation. An Axima Performance MALDI-ToF-ToF (Shimadzu, Manchester, UK) equipped with a 337nm nitrogen laser was used in the analysis. Measurements were performed in reflectron positive mode using an acceleration voltage of 20kV. Red phosphorus was used as the calibrant. Results: A method for direct lipid blood analysis was developed and described as follows. A volume of 10μl of blood was mixed 1:1 v/v with a solution of CH3 OH/ CHCl3 (1:1 v/v). The solution was vortexed and left to separate for 10 minutes, resulting in 6μ l of recoverable solution. After we obtained the blood and made the solution, 10μl of blood was centrifuged for 4 minutes (30k relative centrifugal force) and 4μl of plasma recovered. The plasma was then diluted 1:2 v/v with the CH3 OH-CHCl3 solution. Stirring and extraction were carried out as above and 4μl from the organic phase was collected and subjected to MALDI analysis. THAP (2′ ,4′ ,6′ -Trihydroxyacetophenone) (10mg/ml) was dissolved in 7:3 v/v CH3 OH/H2 O (50mMammonium citrate) and used as the matrix. Samples were mixed 1:1 v/v with the matrix solution and 1μl spotted on the MALDI target. Conclusions: By using only 10μ l of blood, it was possible to obtain a fingerprint of plasma lipids for diagnostic assessment.

NI-10 * A MASS SPECTROMETRY IMAGING PLATFORM TO STUDY DRUG-SUSCEPTIBILITY OF BRAIN TUMORS

Brain tumor heterogeneity poses significant challenges to their study and moreover to their treatment. A tool that could characterize a tumor specimen based on its cellular composition and response to treatment could assist the treating neuro-oncologist in determining the optimal treatment. We have developed a chip to culture dissociated cells in an organized array to categorize cell populations and subject them to targeted therapy. The chip was designed for analysis by optical and mass spectrometry imaging. The microwell array coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is used for the identification of clinical markers of response to targeted therapy. The new microwell array was validated for single-cell imaging by mass spectrometry based on correlation to fluorescence microscopy. Adherent NIH3T3 mouse fibroblasts expressing the enhanced GFP reporter gene and/or U87 cells stained with DAPI were incubated on microwell plates to allow their isolation at the bottom of each well. After cell fixation, the chip was imaged by fluorescence microscopy to determine the total number of wells occupied by cells, and the number of cells per well. The chips were coated with the CHCA matrix and analyzed by MALDI MSI. The statistical analyses of the MALDI MSI data revealed molecular signatures characteristic of each cell type, and mostly assigned to phospholipids. We also monitored the U87 cell response to a targeted inhibitor treatment to identify single-cell barcoding of drug response by MALDI MSI. The platform is further applied to the analysis of dissociated cells from surgical glioma specimens to characterize their heterogeneity and evaluate targeted treatment response. The proposed research has the potential to open ways for more comprehensive diagnostic approaches and to accompany them with direct evaluation of drug effect evaluation for the tailoring of brain cancer treatment.

Laser desorption/ionization mass spectrometric analysis of surfactants on functionalized carbon nanotubes

Recently, unmodified and carboxylated carbon nanotubes have been used as assisting surfaces laser desorption/ionization (LDI) in mass spectrometry. The functionalization of carbon nanotubes with organic compounds should lead to a gamut of other promising LDI-assisting surfaces. Carboxylated carbon nanotubes were functionalized with sinapinic acid either covalently or by creating an ionic macro-complex. Polyether-based surfactants were used as analytes to examine the properties of these new matrices. Mass spectrometric analysis was conducted on a LDI-quadrupole time-of-flight (QTOF) mass spectrometer. Carbon nanotube surfaces were deposited from suspension using the dried-droplet method. The functionalization of the carbon nanotubes was confirmed with Fourier transform infrared (FTIR) spectroscopy. The usefulness of each material was examined with two poly(ethylene glycol) hexadecyl ether amphiphiles (Brij® 52 and Brij® 56) and a poly(ethylene glycol) monomethyl ether as analytes. Generally, the mass spectra obtained with carbon nanotubes covalently functionalized with sinapinic acid as a matrix had peaks with higher intensities than those obtained with carbon nanotubes functionalized by ionic macro-complex formation. The presented new materials based on functionalized carbon nanotubes are effective in the LDI mass analysis of polyether amphiphiles and poly(ethylene glycol) monomethyl ether. This type of assisting surfaces can be highly modified by appropriate functionalization procedures.