Role of mass spectrometry in the study of interactions between amylin and metal ions.

Amylin (islet amyloid polypeptide [IAPP]) is a neuroendocrine hormone synthesized with insulin in the beta cells of pancreatic islets. The two hormones act in different ways: in fact insulin triggers glucose uptake in muscle and liver cells, removing glucose from the bloodstream and making it available for energy use and storage, while amylin regulates glucose homeostasis. Aside these positive physiological aspects, human amyloid polypeptide (hIAPP) readily forms amyloid in vitro. Amyloids are aggregates of proteins and in the human body amyloids are considered responsible of the development of various diseases. These aspects have been widely described and discussed in literature and to give a view of the highly complexity of this biochemical behavior the different physical, chemical, biological and medical aspects are shortly described in this review. It is strongly affected by the presence on metal ions, responsible for or inhibiting the formation of fibrils. Mass spectrometry resulted (and still results) to be a particularly powerful tool to obtain valid and effective experimental data to describe the hIAPP behavior. Aside classical approaches devoted to investigation on metal ion-hIAPP structures, which reflects on the identification of metal-protein interaction site(s) and of possible metal-induced conformational changes of the protein, interesting results have been obtained by ion mobility mass spectrometry, giving, on the basis of collisional cross-section data, information on both the oligomerization processes and the conformation changes. Laser ablation electrospray ionization-ion mobility spectrometry-mass spectrometry (LAESI-IMS-MS), allowed to obtain information on the binding stoichiometry, complex dissociation constant, and the oxidation state of the copper for the amylin-copper interaction. Alternatively to inorganic ions, small organic molecules have been tested by ESI-IMS-MS as inhibitor of amyloid assembly. Also in this case the obtained data demonstrate the validity of the ESI-IMS-MS approach as a high-throughput screen for inhibitors of amyloid assembly, providing valid information concerning the identity of the interacting species, the nature of binding and the effect of the ligand on protein aggregation. Effects of Cu2+ and Zn2+ ions in the degradation of human and murine IAPP by insulin-degrading enzyme were studied by liquid chromatography/mass spectrometry (LC/MS). The literature data show that mass spectrometry is a highly valid and effective tool in the study of the amylin behavior, so to individuate medical strategies to avoid the undesired formation of amyloids in in vivo conditions.

Epithelioid Pleural Mesothelioma Is Characterized by Tertiary Lymphoid Structures in Long Survivors: Results from the MATCH Study.

Pleural mesothelioma (PM) is an aggressive tumor with few therapeutic options. Although patients with epithelioid PM (ePM) survive longer than non-epithelioid PM (non-ePM), heterogeneity of tumor response in ePM is observed. The role of the tumor immune microenvironment (TIME) in the development and progression of PM is currently considered a promising biomarker. A few studies have used high-throughput technologies correlated with TIME evaluation and morphologic and clinical data. This study aimed to identify different morphological, immunohistochemical, and transcriptional profiles that could potentially predict the outcome. A retrospective multicenter cohort of 129 chemonaive PM patients was recruited. Tissue slides were reviewed by dedicated pathologists for histotype classification and immunophenotype of tumor-infiltrating lymphocytes (TILs) and lymphoid aggregates or tertiary lymphoid structures (TLS). ePM (n = 99) survivors were further classified into long (>36 months) or short (<12 months) survivors. RNAseq was performed on a subset of 69 samples. Distinct transcriptional profiling in long and short ePM survivors was found. An inflammatory background with a higher number of B lymphocytes and a prevalence of TLS formations were detected in long compared to short ePM survivors. These results suggest that B cell infiltration could be important in modulating disease aggressiveness, opening a pathway for novel immunotherapeutic approaches.

MASS SPECTROMETRY FOR A HOLISTIC VIEW OF NATURAL EXTRACTS OF PHYTOTHERAPEUTIC INTEREST.

In the study of natural products new strategies which favor a holistic approach, integrating the traditional reductionist methods usually employed, have been proposed. In this frame, the studies carried out by us in the last decade show that fingerprints, mainly obtained by electrospray ionization mass spectrometry (ESI-MS), lead to the characterization of natural extracts from different botanical species but also of phytotherapeutic products constituted by mixtures of extracts from different plants. Laser desorption ionization and matrix-assisted laser desorption ionization techniques were also employed and by the use of different matrices some complementary results were achieved. Results obtained by standard spectrophotometric and liquid chromatography methods were compared with those achieved by direct infusion of the extract in ESI-MS conditions, indicating an excellent agreement between the two approaches. The findings of these researches were considered in the frame of complex systems theory, investigating how relationships between a system's parts can give rise to its collective behaviors and how the system interacts and forms relationships with its environment. In this view, the peculiar pharmacological behavior of biologically active natural compounds can be justified by the occurrence of molecular interactions due to the high complexity of the natural matrix. Some of these interactions have been widely studied in the case of green tea extracts (GTEs) proving unequivocally the presence of caffeine/catechin complexes in GTE samples. The presence of bimolecular complexes has been observed also in the case of Ceylon tea and Mate extracts. These data indicate that the formation of complexes in natural extracts is a common behavior and their presence must be considered in the description of natural extracts and, consequently, in their biological activity. ©2020 John Wiley & Sons Ltd. Mass Spec Rev.

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells.

Membrane proteins constitute around 20-30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of "omics" techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

A method for assessing plasma free fatty acids from C2 to C18 and its application for the early detection of colorectal cancer.

The targeted analysis of free fatty acids (FFAs) is attracting interest since several years with a plenty of studies. However, most of them are devoted to the solely determination of the short-chain fatty acids (SCFAs) arising from the symbiotic gut microbiota metabolism. Recently, the FFAs analysis highlighted changes in the plasma levels of octanoic and decanoic acids (medium-chain fatty acids or MCFAs) may be associated to gastrointestinal diseases, including colorectal cancer (CRC). Then, the simultaneous quantification of both SCFAs and MCFAs could be useful to put in evidence the interconnection between microbiota and metabolic alterations during hosts' disease. To this aim, it was developed an isotopic dilution gas-chromatography coupled mass spectrometry (ID/GC-MS) method for the targeted analysis of both linear and branched FFAs (SCFAs, MCFAs, and LCFAs) in human plasma samples as specific markers for both microbiota and host metabolic alterations. In order to minimize sample manipulation procedures, an efficient, sensible and low time-consuming procedure is presented, which relies in a simple liquid-liquid extraction before the determination of underivatized free acids (FFAs) by Single Ion Monitoring (SIM) acquisition. The reached detection limits (LODs) were less than 100 µg L-1 for most of analytes, except for acetic, hexadecanoic and octadecanoic acids that showed a LOD > 1 mg L-1. Methods accuracy and precision, obtained by the analysis of the FFAs mixtures showed accuracy values between 84% and 100% and precision (RSD %) between 0.1% and 12.4% at the concentration levels tested. The proposed ID/GC-MS method was applied in a case study to evaluate the FFAs as specific markers for both microbiota and host alterations in CRC patients. Obtained results highlight the advantage of present method for its rapidity, simplicity, and robustness.

An investigation on [5 fluorouracil and epigallocatechin-3-gallate] complex activity on HT-29 cell death and its stability in gastrointestinal fluid.

Recently an enhancement of the sensitivity of colorectal cancer (CRC) cells by 5-fluorouracil (5FU) due to the concurrent treatment with epigallocatechin-3-gallate (EGCG) has been found. In the present paper, to investigate on this aspect, adenocarcinoma cells HT29 were treated with 5FU, EGCG and an equimolar mixture of 5FU and EGCG ([5FU+EGCG]) and cell viability was determined. While 5FU exhibits a clear activity, EGCG alone does not express any activity. However by treating the cells with [5FU+EGCG] a strong effect of EGCG is evidenced: the sensitivity of HT29 cells to 5FU was increased by 12-fold. A simulation of the behavior of [5FU+EGCG] in different compartments of the gastrointestinal digestion model was also performed. 5FU and EGCG solubilized into a mixture of digestive fluids analyzed by mass spectrometry did not lead to signals of 5FU, EGCG and the related complex, while by diluting the solution they become detectable. On the contrary, when 5FU and EGCG are submitted to the step-by-step digestion model procedure, the analysis did not show the presence of 5FU, EGCG and [5FU+EGCG]. This behaviour could be ascribed to the instability of these compounds due to the too severe digestion conditions and/or to the complexity of the matrix which could lead in ESI conditions to the suppression of the signals of the analytes of interest.

Mass spectrometry in the study of molecular complexes between 5-fluorouracil and catechins.

5-Fluorouracil (5FU) is a widely employed antineoplastic agent that acts as antimetabolite. However, 5FU activity is strongly reduced against a subset of cancer cells called cancer stem cells (CSCs), which are believed to be responsible for chemoresistance and tumour recurrence. It was found that epigallocatechin-3-gallate (EGCG), the most abundant catechin present in green tea extract, suppresses CSCs grown in various cancers. This chemosensitizing effect of EGCG was investigated in 5FU-resistant (5FUR) CRC cells, showing that EGCG enhances 5FU-induced cytotoxicity. However, the real mechanism of an improved 5FU chemosensitivity in the presence of EGCG was not evaluated. Considering the capability of catechins to form bimolecular noncovalent complexes, in the present study, the interaction of catechins and 5FU was studied by different mass spectrometric approaches. The ESI(+) and ESI(-) spectra of [5FU-catechin] mixtures were studied, showing the formation of protonated and deprotonated bimolecular complexes, whose nature was confirmed by MS/MS experiments (product and precursor ion scans). To exclude the possible origin of these species as ESI artefacts, a further series of experiments were performed by high-resolution liquid chromatography-mass spectrometry. By this approach, bimolecular complexes have been detected at retention times different from those of free 5FU and catechins, proving their presence in the original solution. Analogous studies were performed on 5FU-green tea extract mixtures, showing that 5FU leads to complexes not only with EGCG but also with other catechins. These molecular species, differently to free 5FU drug alone, would in principle possess a new biological activity and could be an explanation of the described activity cited above.

An electrospray ionization study on complexes of amylin with Cu(II) and Cu(I).

Human amylin (hIAPP) is one of a number of different peptides known to be responsible for the formation of amyloid fibrils in the pancreas of subjects with Type 2 diabetes mellitus. It was recognized that metal ions such as Cu(II) are implicated in the aggregation process of amyloidogenic peptides. However, the role of Cu(II) ions in the aggregation and dyshomeostasis of amylin has been controversial. Considering that most of the research reported in the literature pertain to the interactions between Cu(II) and amylin, we thought of interest to compare the interactions of Cu(II) and Cu(I) ions with amylin by electrospray ionization (ESI) mass spectrometry and collisional experiments, to elucidate possible differences in structural aspects of the complexes so formed. The ESI mass spectra of solutions containing hIAPP and Cu(I) or Cu(II) ions show the formation of hIAPP-Cu complexes. In both cases, M + Cu ions with three and four positive charges are detected. However, a series of fragment ions, absent in the ESI spectrum of untreated hIAPP, become detectable. Some of them are common for both Cu(I) and Cu(II) complexes, whereas others are specific for the complexes containing Cu in different oxidation states. Some fragments imply the involvement of residues His18, Ser19, Ser20, Asn21, and Asn22 in the complex formation, but the detection of the fragment b22 3+ indicates the presence of copper ions in a different position. This suggests different interaction sites between Cu(II) and Cu(I) and hIAPP. In contrast to Cu(II) complex, in the Cu(I) complex, some peculiar structures are present, corresponding to the cleavage of Asn-Asn peptidic bond and to [b30 + Cu(I)]4+ and [b28 + Cu(I)]4+ species. These results are in agreement with the coordination vacancy in [Cu(I)-(peptide)] species, which promotes Cu(I) interaction with additional neighboring donors (mainly N-histidine, and also S-methionine or other groups depending on the peptide conformation) through formation of trigonal T-shaped intermediates.

Evidence of noncovalent complexes in some natural extracts: Ceylon tea and mate extracts.

Considering the high complexity of natural extracts, because of the presence of organic molecules of different chemical nature, the possibility of formation of noncovalent complexes should be taken into account. In a previous investigation, the formation of bimolecular complexes between caffeine and catechins in green tea extracts (GTE) has been experimentally proven by means of mass spectrometric and 1 H nuclear magnetic resonance experiments. The same approaches have been employed in the present study to evaluate the presence of bimolecular complexes in Ceylon tea and mate extracts. The obtained results show that in the case of Ceylon tea extracts, protonated theaflavin is detectable, together with theaflavin/caffein complexes, while caffeine/catechin complexes, already detected in green tea, are still present but at lower concentration. This aspect is evidenced by the comparison of precursor ion scans performed on protonated caffeine for the two extracts. The spectra obtained in these conditions for GTE and Ceylon tea show that the complexes of caffeine with epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG), highy abundant in the case of GTE (signal-to-chemical noise ratio in the range 50-100), are negligible (signal-to-chemical noise ratio in the range 2-3) in the case of Ceylon tea. Mate extracts show the formation of bimolecular complexes involving caffeine but not catechins, and chlorogenic acid becomes responsible for other complex formation. Under positive ion and negative ion conditions, accurate mass measurements allow the identification of malealdehyde, chlorogenic acid, caffeine, two isomers of dicaffeoylquinic acid, rutin, and kaempferol-3-O-rutinoside. These data indicate that the formation of complexes in natural extracts is a common behavior, and their presence must be considered in the description of natural extracts and, consequently, in their biological activity.