Proteomic Investigation of Immune Checkpoints and Some of Their Inhibitors.

Immune checkpoints are crucial molecules for the maintenance of antitumor immune responses. The activation or inhibition of these molecules is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals to the various components of the immune system. Over the last 10 years, the inhibition of immune checkpoints, such as cytotoxic T lymphocyte antigen-4, programmed cell death-1, and programmed cell death ligand-1, has taken a leading role in immune therapy. This relatively recent therapy regime is based on the use of checkpoint inhibitors, which enhance the immune response towards various forms of cancer. For a subset of patients with specific forms of cancer, these inhibitors can induce a durable response to therapy; however, the medium response rate to such therapy remains relatively poor. Recent research activities have demonstrated that the disease response to this highly promising therapy resembles the response of many forms of cancer to chemotherapy, where an encouraging initial response is followed by acquired resistance to treatment and progress of the disease. That said, these inhibitors are now used as single agents or in combination with chemotherapies as first or second lines of treatment for about 50 types of cancer. The prevailing opinion regarding immune therapy suggests that for this approach of therapy to deliver on its promise, a number of challenges have to be circumvented. These challenges include understanding the resistance mechanisms to immune checkpoint blockade, the identification of more efficient inhibitors, extending their therapeutic benefits to a wider audience of cancer patients, better management of immune-related adverse side effects, and, more urgently the identification of biomarkers, which would help treating oncologists in the identification of patients who are likely to respond positively to the immune therapies and, last but not least, the prices of therapy which can be afforded by the highest number of patients. Numerous studies have demonstrated that understanding the interaction between these checkpoints and the immune system is essential for the development of efficient checkpoint inhibitors and improved immune therapies. In the present text, we discuss some of these checkpoints, their inhibitors, and some works in which mass spectrometry-based proteomic analyses were applied.

Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins.

Drug resistance remains one of the main causes of poor outcome in cancer therapy. It is also becoming evident that drug resistance to both chemotherapy and to antibiotics is driven by more than one mechanism. So far, there are at least eight recognized mechanisms behind such resistance. In this review, we choose to discuss one of these mechanisms, which is known to be partially driven by a class of transmembrane proteins known as ATP-binding cassette (ABC) transporters. In normal tissues, ABC transporters protect the cells from the toxic effects of xenobiotics, whereas in tumor cells, they reduce the intracellular concentrations of anticancer drugs, which ultimately leads to the emergence of multidrug resistance (MDR). A deeper understanding of the structures and the biology of these proteins is central to current efforts to circumvent resistance to both chemotherapy, targeted therapy, and antibiotics. Understanding the biology and the function of these proteins requires detailed structural and conformational information for this class of membrane proteins. For many years, such structural information has been mainly provided by X-ray crystallography and cryo-electron microscopy. More recently, mass spectrometry-based methods assumed an important role in the area of structural and conformational characterization of this class of proteins. The contribution of this technique to structural biology has been enhanced by its combination with liquid chromatography and ion mobility, as well as more refined labelling protocols and the use of more efficient fragmentation methods, which allow the detection and localization of labile post-translational modifications. In this review, we discuss the contribution of mass spectrometry to efforts to characterize some members of the ATP-binding cassette (ABC) proteins and why such a contribution is relevant to efforts to clarify the link between the overexpression of these proteins and the most widespread mechanism of chemoresistance.

Multiplexed MRM-based proteomics for identification of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2-4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin resistance, and chronic hyperglycaemia. The aim of this investigation was to assess, by a multimarker mass spectrometry approach, the predictive role of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus. METHODS: The study considered 34 patients with both T2DM and CHD, 31 patients with T2DM and without CHD, and 30 patients without diabetes with a diagnosis of CHD. Plasma samples of subjects were analysed through a multiplexed targeted liquid chromatography mass spectrometry (LC-MS)-based assay, namely Multiple Reaction Monitoring (MRM), allowing the simultaneous detection of peptides derived from a protein of interest. Gene Ontology (GO) Analysis was employed to identify enriched GO terms in the biological process, molecular function, or cellular component categories. Non-parametric multivariate methods were used to classify samples from patients and evaluate the relevance of the analysed proteins' panel. RESULTS: A total of 81 proteins were successfully quantified in the human plasma samples. Gene Ontology analysis assessed terms related to blood microparticles, extracellular exosomes and collagen-containing extracellular matrix. Preliminary evaluation using analysis of variance (ANOVA) of the differences in the proteomic profile among patient groups identified 13 out of the 81 proteins as significantly different. Multivariate analysis, including cluster analysis and principal component analysis, identified relevant grouping of the 13 proteins. The first main cluster comprises apolipoprotein C-III, apolipoprotein C-II, apolipoprotein A-IV, retinol-binding protein 4, lysozyme C and cystatin-C; the second one includes, albeit with sub-grouping, alpha 2 macroglobulin, afamin, kininogen 1, vitronectin, vitamin K-dependent protein S, complement factor B and mannan-binding lectin serine protease 2. Receiver operating characteristic (ROC) curves obtained with the 13 selected proteins using a nominal logistic regression indicated a significant overall distinction (p < 0.001) among the three groups of subjects, with area under the ROC curve (AUC) ranging 0.91-0.97, and sensitivity and specificity ranging from 85 to 100%. CONCLUSIONS: Targeted mass spectrometry approach indicated 13 multiple circulating proteins as possible biomarkers of cardiovascular damage progression associated with T2DM, with excellent classification results in terms of sensitivity and specificity.

Mass Spectrometry-Based Proteomics: Analyses Related to Drug-Resistance and Disease Biomarkers.

Mass spectrometry-based proteomics is a key player in research efforts to characterize aberrant epigenetic alterations, including histone post-translational modifications and DNA methylation. Data generated by this approach complements and enrich datasets generated by genomic, epigenetic and transcriptomics approaches. These combined datasets can provide much-needed information on various mechanisms responsible for drug resistance, the discovery and validation of potential biomarkers for different diseases, the identification of signaling pathways, and genes and enzymes to be targeted by future therapies. The increasing use of high-resolution, high-accuracy mass spectrometers combined with more refined protein labeling and enrichment procedures enhanced the role of this approach in the investigation of these epigenetic modifications. In this review, we discuss recent MS-based studies, which are contributing to current research efforts to understand certain mechanisms behind drug resistance to therapy. We also discuss how these MS-based analyses are contributing to biomarkers discovery and validation.

Mass Spectrometry Contribution to Pediatric Cancers Research.

For over four decades, mass spectrometry-based methods have provided a wealth of information relevant to various challenges in the field of cancers research. These challenges included identification and validation of novel biomarkers for various diseases, in particular for various forms of cancer. These biomarkers serve various objectives including monitoring patient response to the various forms of therapy, differentiating subgroups of the same type of cancer, and providing proteomic data to complement datasets generated by genomic, epigenetic, and transcriptomic methods. The same proteomic data can be used to provide prognostic information and could guide scientists and medics to new and innovative targeted therapies The past decade has seen a rapid emergence of epigenetics as a major contributor to carcinogenesis. This development has given a fresh momentum to MS-based proteomics, which demonstrated to be an unrivalled tool for the analyses of protein post-translational modifications associated with chromatin modifications. In particular, high-resolution mass spectrometry has been recently used for systematic quantification of chromatin modifications. Data generated by this approach are central in the search for new therapies for various forms of cancer and will help in attempts to decipher antitumor drug resistance. To appreciate the contribution of mass spectrometry-based proteomics to biomarkers discovery and to our understanding of mechanisms behind the initiation and progression of various forms of cancer, a number of recent investigations are discussed. These investigations also include results provided by two-dimensional gel electrophoresis combined with mass spectrometry.

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.

Mass Spectrometry Study about In Vitro and In Vivo Reaction between Metformin and Glucose: A Preliminary Investigation on Alternative Biological Behavior.

Metformin is the most prescribed glucose-lowering drug worldwide; globally, over 100 million patients are prescribed this drug annually. Some different action mechanisms have been proposed for this drug, but, surprisingly, no metabolite of metformin has ever been described. It was considered interesting to investigate the possible reaction of metformin with glucose following the Maillard reaction pattern. The reaction was first performed in in vitro conditions, showing the formation of two adducts that originated by the condensation of the two molecular species with the losses of one or two water molecules. Their structures were investigated by liquid chromatography coupled with mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS) and accurate mass measurements (HRMS). The species originated via the reaction of glucose and metformin and were called metformose and dehydrometformose, and some structural hypotheses were conducted. It is worth to emphasize that they were detected in urine samples from a diabetic patient treated with metformin and consequently they must be considered metabolites of the drug, which has never been identified before now. The glucose-related substructure of these compounds could reflect an improved transfer across cell membranes and, consequently, new hypotheses could be made about the biological targets of metformin.

Proteomic Approaches in the Study of Placenta of Pregnancy Complicated by Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM), a glucose intolerance developing or first recognized during pregnancy, leads to a series of short- and long-term maternal and fetal complications, somehow related to placenta structural and functional changes. The focus and the objective of the present review are to discuss the results which can be obtained by different mass spectrometric approaches in the study of placenta protein profile. Thus, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) has been applied on placenta omogenates before and after one-dimensional electrophoretic separation, followed by tryptic digestion. MALDI imaging was used for direct investigation on the placenta tissue (both maternal and fetal sides). The results showed that some differences among the absolute abundances of some proteins are present for placenta samples from GDM patients. The majority of investigations were carried out by two-dimensional electrophoresis (2DE) followed by LC-MS/MS or, directly by the label-free LC-MSE approach. It should be emphasized that all these techniques were showed differences in the protein expression between the placenta samples from healthy or GDM subjects. 2DE was also employed to separate and compare placental protein levels from GDM and the control groups: differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and were further confirmed by Western blotting. The physiopathological significance of the obtained results are reported and discussed in this narrative review. The experimental data obtained until now show that the newest, mass spectrometric approaches can be considered a valid tool to investigate the possible changes of placenta in the presence of GDM.

q value and parent energy optimization using a low-voltage ionization approach increases resolution in linear ion trap mass spectrometry.

In this work, the isolation step in the linear ion trap was performed using different "q values" conditions at a low collision-induced dissociation (CID) energy leading to the parent ion resolution improvements, reasonably due to better ion energy distribution. According to the results, we obtained a greater resolution and mass accuracy operating in both traditional electrospray and low voltage ionization near the q value = 0.778 and with a CID energy of 10%. This effect was evaluated with low-molecular-mass compounds (skatole and arginine). The proposed optimization yielded a superior instrument performance without adding technological complexity to mass spectrometry analyses.

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.

In silico evaluation of the interaction between ACE2 and SARS-CoV-2 Spike protein in a hyperglycemic environment.

The worse outcome of COVID-19 in people with diabetes mellitus could be related to the non-enzymatic glycation of human ACE2, leading to a more susceptible interaction with virus Spike protein. We aimed to evaluate, through a computational approach, the interaction between human ACE2 receptor and SARS-CoV-2 Spike protein under different conditions of hyperglycemic environment. A computational analysis was performed, based on the X-ray crystallographic structure of the Spike Receptor-Binding Domain (RBD)-ACE2 system. The possible scenarios of lysine aminoacid residues on surface transformed by glycation were considered: (1) on ACE2 receptor; (2) on Spike protein; (3) on both ACE2 receptor and Spike protein. In comparison to the native condition, the number of polar bonds (comprising both hydrogen bonds and salt bridges) in the poses considered are 10, 6, 6, and 4 for the states ACE2/Spike both native, ACE2 native/Spike glycated, ACE2 glycated/Spike native, ACE2/Spike both glycated, respectively. The analysis highlighted also how the number of non-polar contacts (in this case, van der Waals and aromatic interactions) significantly decreases when the lysine aminoacid residues undergo glycation. Following non-enzymatic glycation, the number of interactions between human ACE2 receptor and SARS-CoV-2 Spike protein is decreased in comparison to the unmodified model. The reduced affinity of the Spike protein for ACE2 receptor in case of non-enzymatic glycation may shift the virus to multiple alternative entry routes.

ELISA based on peptide antigens reproducing cross-reactive viral epitopes to detect antibodies in latent autoimmune diabetes in adults vs. type 1 diabetes.

Diagnosis of Latent Autoimmune Diabetes in Adults (LADA) is based on the adult-age, anti-islet autoantibodies, and temporary insulin-independence. As in Type-1-Diabetes (T1DM), autoimmunity may trigger LADA and enteroviruses-infections can play a role. Anti-human Glutamic-Acid-Decarboxylase (hGAD) autoantibodies are accepted clinical biomarkers, but do not discriminate LADA vs. T1DM. The hypothesis is that protein antigens detecting anti-hGAD antibodies do not expose epitopes specific for different disease forms. We investigated the diagnostic value of autoantibodies in LADA vs. T1DM to peptides of hGAD65/67 isoforms, and Enterovirus-Coxsackie-B4 (CVB4), as antigens sharing the epitope PEVKXK (X: E/T) included in CD8 T-cell CVB4 epitope restricted by diabetes-associated HLA-A2.1. Statistically significant differences of IgM and/or IgG in LADA and T1DM vs. controls were identified. In LADA IgMs to GAD65/67 peptides are diagnostics, IgGs to GAD65/67 peptides correlate with anti-CVB4 peptide antibodies. IgM and/or IgG to all tested peptides can predict LADA, monitoring CVB4 infected patients, improving LADA vs. T1DM stratification.•A customized SP-ELISA based on synthetic peptides Ac-hGAD65(250-273)-NH2 (1), Ac-hGAD67(258-281)-NH2 (2), and Ac-CVB4P2C(28-50)-NH2 (3) is described.•The method was designed to detect specific IgM and/or IgG in LADA, T1DM, vs. controls•Final aim is improvement of LADA vs. T1DM patient stratification.

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.

Cross-reactive peptide epitopes of Enterovirus Coxsackie B4 and human glutamic acid decarboxylase detecting antibodies in latent autoimmune diabetes in adults versus type 1 diabetes.

BACKGROUND: Diagnosis of latent autoimmune diabetes in adults (LADA) is usually based on the adult age, anti-pancreatic islet cell antibodies detection, and insulin independence. This study investigates the diagnostic value of antibodies against human glutamic acid decarboxylase (hGAD) peptides in LADA and type 1 diabetes mellitus (T1DM) patients, and their cross-reactivity with an Enterovirus Coxsackie B4 (CVB4) shared epitope. METHODS: Sera from 27 LADA patients, 23 T1DM patients, and 24 controls were tested in ELISA for antibodies against hGAD peptides and a selected sequence of P2C protein of CVB4 (CVB4P2C). Diagnostic power of peptides was analyzed by ROC-curve analysis and cross-reactivity among peptides evaluated. RESULTS: IgM and IgG antibodies showed significant differences between LADA and T1DM versus controls for all peptides. Antibody responses present high agreement among peptides for IgM and IgG-isotypes in T1DM, which is not reproduced in LADA. IgM antibodies showed high predicting diagnostic power particularly in LADA (sensitivity > 85%, specificity 95.8%). CONCLUSIONS: Our study highlights the usefulness of peptides as diagnostic antigens in T1DM and LADA, and extends previous findings by comparing IgM and IgG-isotype antibodies in the same population. Additionally, results highlight the role of the entourage in the shared sequon PEVKXK in GAD and CVB4P2C particularly in IgMs identification.

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.

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.

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.

Is the placental proteome impaired in well-controlled gestational diabetes?

In pregnancy complicated by gestational diabetes mellitus (GDM), the human placenta shows several pathological functional and structural changes, but the extent to which maternal glycemic control contributes to placental abnormalities remains unclear. The aim of this study was to profile and compare the proteome of placentas from healthy pregnant women and those with GDM, to investigate the placenta-specific protein composition and possible changes of its function in presence of GDM. Quantitative proteomic analysis, based on LC-MSE approach, revealed that higher (approximately 15% increase) levels of galectin 1 and collagen alpha-1 XIV chain (although the difference regarding the latter was at the limit of significance) were present in GDM samples, while heat shock 70 kDa protein 1A/1B was less abundant in GDM placental tissue. These data seem to indicate that GDM, when well controlled, did not markedly affect the placental proteome.

New Mass Spectrometric Approaches for the Quantitative Evaluation of Anticancer Drug Levels in Treated Patients.

Alternatively to the well-consolidated liquid chromatography coupled to tandem mass spectrometry approach used for the evaluation of anticancer drug concentrations in treated patients, new mass spectrometric methods have been proposed and tested recently. They exhibited faster analysis time and, at first sight, simpler instrumental approaches. However, results obtained by these methods require an in-depth evaluation, because of their strong dependence on the experimental set-up. In this short review, the quantification of irinotecan, sunitinib, and 6-α-hydroxy paclitaxel (the main metabolite of paclitaxel) by laser desorption ionization techniques (matrix-assisted laser desorption/ionization, nanostructure-assisted laser desorption/ionization, and surface-assisted laser desorption/ionization) is reported and discussed, showing the advantages but also the drawbacks of the methods. The matrix-assisted laser desorption/ionization approach led to the most reliable results, and the cross-validation for the quantitative analysis of irinotecan indicates that this method can be fruitfully used for therapeutic drug monitoring and pharmacokinetic studies. Another recently proposed technique, paper spray mass spectrometry, has been tested for the quantitative measurement of imatinib in plasma samples. Even if the approach is, at first sight, really simple, the parameterization of the analytical and instrumental aspects has required many efforts to reach satisfactory results. What it should be expected in the future is the evaluation of these methods, not only in scientific environments dedicated to instrument development, but also in clinical chemistry laboratories, to evaluate their effectiveness and to give new and valid tools for TDM and for other qualitative or quantitative measurements of biomedical interest.