Intact cell mass spectrometry coupled with machine learning reveals minute changes induced by single gene silencing.

Intact (whole) cell MALDI TOF mass spectrometry is a commonly used tool in clinical microbiology for several decades. Recently it was introduced to analysis of eukaryotic cells, including cancer and stem cells. Besides targeted metabolomic and proteomic applications, the intact cell MALDI TOF mass spectrometry provides a sufficient sensitivity and specificity to discriminate cell types, isogenous cell lines or even the metabolic states. This makes the intact cell MALDI TOF mass spectrometry a promising tool for quality control in advanced cell cultures with a potential to reveal batch-to-batch variation, aberrant clones, or unwanted shifts in cell phenotype. However, cellular alterations induced by change in expression of a single gene has not been addressed by intact cell mass spectrometry yet. In this work we used a well-characterized human ovarian cancer cell line SKOV3 with silenced expression of a tumor suppressor candidate 3 gene (TUSC3). TUSC3 is involved in co-translational N-glycosylation of proteins with well-known global impact on cell phenotype. Altogether, this experimental design represents a highly suitable model for optimization of intact cell mass spectrometry and analysis of spectral data. Here we investigated five machine learning algorithms (k-nearest neighbors, decision tree, random forest, partial least squares discrimination, and artificial neural network) and optimized their performance either in pure populations or in two-component mixtures composed of cells with normal or silenced expression of TUSC3. All five algorithms reached accuracy over 90 % and were able to reveal even subtle changes in mass spectra corresponding to alterations of TUSC3 expression. In summary, we demonstrate that spectral fingerprints generated by intact cell MALDI-TOF mass spectrometry coupled to a machine learning classifier can reveal minute changes induced by alteration of a single gene, and therefore contribute to the portfolio of quality control applications in routine cell and tissue cultures.

Pulsed laser ablation synthesis of fresh Te nanoparticles for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) applications.

This work aims to demonstrate the potential of pulsed laser ablation synthesis (PLA) of tellurium nanoparticles (Te NPs) for use in matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) applications. An experimental laboratory setup for PLA synthesis of fresh Te NPs was designed to prevent unwanted aggregation of uncoated Te NPs and avoid the need to use additional modifiers. Performing pulsed laser ablation synthesis in liquid (PLAL) using acetone was found to be the optimal way of preparing Te NPs. Another possibility is to use commercially available laser ablation devices for laser ablation - inductively coupled plasma mass spectrometry (LA-ICP-MS) to perform PLA in a helium atmosphere, but this approach is less efficient and results in the formation of unwanted larger particles. The prepared Te NPs were studied using the transmission electron microscopy (TEM) and dynamic light scattering (DLS) methods. TEM images showed the formation of Te NP nanochains composed of many crystallized Te NPs with sizes ranging from 8 to 15 nm. The various size distributions of the synthesized Te NPs identified using the DLS method correspond to the size distributions of aggregations rather than individual Te NPs. The synthesized Te NPs were used for a pilot study of their possible use with the MALDI-MS technique. An important effect was observed when Te NPs were used to perform a MALDI-MS analysis of the α-cyclodextrin (α-CD) and cucurbit[7]uril (CB7) macrocycles, which consisted in a decline in the formation of matrix adducts. Furthermore, several changes in MALDI-MS mass spectra of intact cells and a positive effect of Te NPs on the crystallization of the MALDI-MS matrix were observed.

Improved Screening of Monoclonal Gammopathy Patients by MALDI-TOF Mass Spectrometry.

Monoclonal gammopathies are a group of blood diseases characterized by presence of abnormal immunoglobulins in peripheral blood and/or urine of patients. Multiple myeloma and plasma cell leukemia are monoclonal gammopathies with unclear etiology, caused by malignant transformation of bone marrow plasma cells. Mass spectrometry with matrix-assisted laser desorption/ionization and time-of-flight detection is commonly used for investigation of the peptidome and small proteome of blood plasma with high accuracy, robustness, and cost-effectivity. In addition, mass spectrometry coupled with advanced statistics can be used for molecular profiling, classification, and diagnosis of liquid biopsies and tissue specimens in various malignancies. Despite the fact there have been fully optimized protocols for mass spectrometry of normal blood plasma available for decades, in monoclonal gammopathy patients, the massive alterations of biophysical and biochemical parameters of peripheral blood plasma often limit the mass spectrometry measurements. In this paper, we present a new two-step extraction protocol and demonstrated the enhanced resolution and intensity (>50×) of mass spectra obtained from extracts of peripheral blood plasma from monoclonal gammopathy patients. When coupled with advanced statistics and machine learning, the mass spectra profiles enabled the direct identification, classification, and discrimination of multiple myeloma and plasma cell leukemia patients with high accuracy and precision. A model based on PLS-DA achieved the best performance with 71.5% accuracy (95% confidence interval, CI = 57.1-83.3%) when the 10× repeated 5-fold CV was performed. In summary, the two-step extraction protocol improved the analysis of monoclonal gammopathy peripheral blood plasma samples by mass spectrometry and provided a tool for addressing the complex molecular etiology of monoclonal gammopathies.

A novel heteroleptic Cu(II)-phenanthroline-UDCA complex as lipoxygenase inhibitor and ER-stress inducer in cancer cell lines.

A new heteroleptic copper(II) compound named C0-UDCA was prepared by reaction of [Cu(phen)2(OH2)](ClO4)2 (C0) with the bile ursodeoxycholic acid (UDCA). The resulting compound is able to inhibit the lipoxygenase enzyme showing more efficacy than the precursors C0 and UDCA. Molecular docking simulations clarified the interactions with the enzyme as due to allosteric modulation. The new complex shows antitumoral effect on ovarian (SKOV-3) and pancreatic (PANC-1) cancer cells at the Endoplasmic Reticulum (ER) level by activating the Unfolded Protein Response. In particular, the chaperone BiP, the pro-apoptotic protein CHOP and the transcription factor ATF6 are upregulated in the presence of C0-UDCA. The combination of Intact Cell MALDI-MS and statistical analysis have allowed us to discriminate between untreated and treated cells based on their mass spectrometry fingerprints.

Laser ablation synthesis of tellurium carbides using tellurium with nanodiamond nanocomposite.

RATIONALE: Carbides, including tellurium carbides (TeC), play crucial roles in diverse applications, but TeC synthesis has not been described in the literature. Laser ablation synthesis (LAS) coupled with mass spectrometry was used here for in situ TeC clusters synthesis and identification of the reaction products to better understand TeC formation. METHODS: Laser desorption ionization time-of-flight mass spectrometry (LDI-TOFMS) was used to generate the TeC clusters and determine their stoichiometry via computer modeling of isotopic patterns. RESULTS: A simple one-pot procedure was developed for Te-nanodiamond nanocomposite preparation. A suspension of fine-powdered Te was mixed with a suspension of nanodiamonds (both in acetonitrile), and the resulting precipitated nanocomposite was suitable for the synthesis of TemCn clusters using LDI. Various unary and binary clusters were formed. The stoichiometry of the novel TemCn clusters, determined via computer modeling of isotopic patterns, is reported here for the first time. CONCLUSIONS: The Te-nanodiamond composite was found to be the most suitable precursor for the generation of TemCn clusters. In total, 35 binary TemCn clusters were identified, when several of them were not obtained using commercial TeC material.

Artificial Neural Networks Coupled with MALDI-TOF MS Serum Fingerprinting To Classify and Diagnose Pathological Pain Subtypes in Preclinical Models.

Pathological pain subtypes can be classified as either neuropathic pain, caused by a somatosensory nervous system lesion or disease, or nociplastic pain, which develops without evidence of somatosensory system damage. Since there is no gold standard for the diagnosis of pathological pain subtypes, the proper classification of individual patients is currently an unmet challenge for clinicians. While the determination of specific biomarkers for each condition by current biochemical techniques is a complex task, the use of multimolecular techniques, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), combined with artificial intelligence allows specific fingerprints for pathological pain-subtypes to be obtained, which may be useful for diagnosis. We analyzed whether the information provided by the mass spectra of serum samples of four experimental models of neuropathic and nociplastic pain combined with their functional pain outcomes could enable pathological pain subtype classification by artificial neural networks. As a result, a simple and innovative clinical decision support method has been developed that combines MALDI-TOF MS serum spectra and pain evaluation with its subsequent data analysis by artificial neural networks and allows the identification and classification of pathological pain subtypes in experimental models with a high level of specificity.

Expandable Lung Epithelium Differentiated from Human Embryonic Stem Cells.

BACKGROUND: The progenitors to lung airway epithelium that are capable of long-term propagation may represent an attractive source of cells for cell-based therapies, disease modeling, toxicity testing, and others. Principally, there are two main options for obtaining lung epithelial progenitors: (i) direct isolation of endogenous progenitors from human lungs and (ii) in vitro differentiation from some other cell type. The prime candidates for the second approach are pluripotent stem cells, which may provide autologous and/or allogeneic cell resource in clinically relevant quality and quantity. METHODS: By exploiting the differentiation potential of human embryonic stem cells (hESC), here we derived expandable lung epithelium (ELEP) and established culture conditions for their long-term propagation (more than 6 months) in a monolayer culture without a need of 3D culture conditions and/or cell sorting steps, which minimizes potential variability of the outcome. RESULTS: These hESC-derived ELEP express NK2 Homeobox 1 (NKX2.1), a marker of early lung epithelial lineage, display properties of cells in early stages of surfactant production and are able to differentiate to cells exhibitting molecular and morphological characteristics of both respiratory epithelium of airway and alveolar regions. CONCLUSION: Expandable lung epithelium thus offer a stable, convenient, easily scalable and high-yielding cell source for applications in biomedicine.

Matrix enrichment by black phosphorus improves ionization and reproducibility of mass spectrometry of intact cells, peptides, and amino acids.

Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.

Laser ablation synthesis of metal-doped gold clusters from composites of gold nanoparticles with metal organic frameworks.

Metal-doped gold clusters, mainly cages, are receiving rapidly increasing attention due to their tunable catalytic properties. Their synthesis is mostly based on complex procedures, including several steps. In this work, via adsorption of gold nanoparticles (AuNPs) from aqueous solution to MOF (metal organic frameworks) of M = Co, Cu, Ni, and Zn with various linkers the {AuNPs, MOF} composites were prepared. These composites were used for laser ablation synthesis (LAS) using a common mass spectrometer. Several series of positively and negatively charged AumMn+/- clusters were observed in mass spectra and their stoichiometry (m = 1-35, n = 1-5) was determined. For each dopant (Co, Cu, Ni, and Zn) ~ 50 different clusters were identified in positive, as well as in negative ion modes. About 100 of these clusters were proposed to be endohedral metal-doped gold cages (for m > 12). The developed approach represents a simple procedure for generating metal-doped gold clusters or endohedral metal-doped gold cages materials with potential applications in medicine and/or electronics.

{Nano-gold, iron(III)-1,3,5-benzene tricarboxylate metal organic framework (MOF)} nano-composite as precursor for laser ablation generation of gold-iron Aum Fen +/- (m = 1-35, n = 1-5) clusters. Mass spectrometric study.

RATIONALE: Gold-iron bimetallic materials have applications in many fields, especially in nanotechnology and biomedicine. The chemistry of iron-doped gold clusters is still not fully understood but opens up the possibility of developing new materials, e.g. of gold cages doped with iron atoms. There have been several theoretical studies on these clusters but only a few experimental studies. METHODS: Laser desorption ionisation (LDI) was used for the generation of Au-Fe bimetallic clusters via laser ablation (337 nm nitrogen laser) of the synthesised nano-composite {nano-gold; Fe(III) 1,3,5-benzene tricarboxylate}, i.e. {AuNPs, Fe-MOF}, while a quadrupole ion trap time-of-flight mass spectrometer, equipped with a reflectron, was used to acquire mass spectra. RESULTS: A {AuNPs, Fe-MOF} nano-composite was prepared and found suitable for the LDI generation of Aum Fen clusters. In addition to Aum +/- (m = 1-35) clusters, a series of positively and negatively charged gold-iron Aum Fen +/- clusters were generated. The mass spectra exhibited evidence for the clusters containing up to five iron atoms. In total, 113 binary Aum Fen +/- clusters (m = 1-35, n = 1-5) were identified in the gas phase. CONCLUSIONS: A synthesised {AuNPs, iron(III)-1,3,5-benzene tricarboxylate MOF} nano-composite was found suitable for the generation of many new gold-iron clusters and mass spectrometry was shown to be an efficient technique for the determination of the cluster stoichiometry. A broad series of over 100 bimetallic Aum Fen clusters, some of them suggested to be gold cages doped with iron atoms (for m = 12 and higher), not only demonstrate a rich and complex chemistry, but also open wide possibilities of biomedical applications.

Mass spectrometric investigation of amorphous Ga-Sb-Se thin films.

Amorphous chalcogenide thin films are widely studied due to their enhanced properties and extensive applications. Here, we have studied amorphous Ga-Sb-Se chalcogenide thin films prepared by magnetron co-sputtering, via laser ablation quadrupole ion trap time-of-flight mass spectrometry. Furthermore, the stoichiometry of the generated clusters was determined which gives information about individual species present in the plasma plume originating from the interaction of amorphous chalcogenides with high energy laser pulses. Seven different compositions of thin films (Ga content 7.6-31.7 at. %, Sb content 5.2-31.2 at. %, Se content 61.2-63.3 at. %) were studied and in each case about ~50 different clusters were identified in positive and ~20-30 clusters in negative ion mode. Assuming that polymers can influence the laser desorption (laser ablation) process, we have used parafilm as a material to reduce the destruction of the amorphous network structure and/or promote the laser ablation synthesis of heavier species from those of lower mass. In this case, many new and higher mass clusters were identified. The maximum number of (40) new clusters was detected for the Ga-Sb-Se thin film containing the highest amount of antimony (31.2 at. %). This approach opens new possibilities for laser desorption ionization/laser ablation study of other materials. Finally, for selected binary and ternary clusters, their structure was calculated by using density functional theory optimization procedure.

Gallium selenide clusters generated via laser desorption ionisation quadrupole ion trap time-of-flight mass spectrometry.

RATIONALE: Gallium selenide thin films important for electronics and phase-change materials are prepared via pulsed laser deposition (PLD); however, there are no studies concerning the analysis of gallium selenide clusters formed in the gas phase. Laser desorption ionisation (LDI) combined with time-of-flight mass spectrometry (TOF-MS) has great potential to generate charged Gam Sen clusters, to analyse them and thus to develop new materials. METHODS: LDI of Ga-Se mixtures using a pulsed laser (337 nm nitrogen) was used to generate gallium-selenide clusters. Mass spectra were recorded (in positive and negative ion mode) on a TOF mass spectrometer equipped with a quadrupole ion trap and reflectron mass analyser. RESULTS: Ga-Se mixtures were found to be suitable for laser ablation synthesis (LAS) of gallium selenide clusters, although their composition was strongly dependent on the laser energy. The effect of laser energy on the stoichiometry of the generated clusters was established. In total, over 100 gallium selenide Gam Sen clusters were generated and identified from Ga-Se mixtures. LDI of Ga2 Se3 crystals showed almost the same clusters up to m/z 1000 with lower intensities, whereas no clusters from Ga2 Se3 were observed above m/z 1000. CONCLUSIONS: A family of over 100 gallium selenide clusters, generated and identified for the first time, shows rich and complex chemistry. Some of the clusters represent new compounds that have the potential to be used in the development of advanced materials.