Marina Naldi

Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodological approach.

Extensive evidence suggests that the self-assembly of amyloid-beta peptide (Aβ) is a nucleation-dependent process that involves the formation of several oligomeric intermediates. Despite neuronal toxicity being recently related to Aβ soluble oligomers, results from aggregation studies are often controversial, mainly because of the low reproducibility of several experimental protocols. Here a multimethodological study that included atomic force microscopy (AFM), transmission electron microscopy (TEM), fluorescence microscopy (FLM), mass spectrometry techniques (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] and electrospray ionization quadrupole time-of-flight [ESI-QTOF]), and direct thioflavin T (ThT) fluorescence spectroscopy were enabled to set up a reliable and highly reproducible experimental protocol for the characterization of the morphology and dimension of Aβ 1-42 (Aβ42) aggregates along the self-assembly pathway. This multimethodological approach allowed elucidating the diverse assembly species formed during the Aβ aggregation process and was applied to the detailed investigation of the mechanism of Aβ42 inhibition by myricetin. In particular, a very striking result was the molecular weight determination of the initial oligomeric nuclei by MALDI-TOF, composed of up to 10 monomers, and their morphology by AFM.

Posttranscriptional changes of serum albumin: Clinical and prognostic significance in hospitalized patients with cirrhosis

Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty‐eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high‐performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child‐Pugh and Model for End‐Stage Liver Disease scores. In hospitalized patients, oxidized and N‐terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N‐terminal truncated isoforms were predictors of 1‐year survival, with greater prognostic accuracy than total serum albumin concentration. Conclusions: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the “effective albumin concentration,” which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity. (Hepatology 2014;60:1850–1859)

UHPLC determination of catechins for the quality control of green tea.

An ultra-high performance liquid chromatography (UHPLC) with UV detection method was developed for the fast quantitation of the most represented and biologically important green tea catechins and caffeine. UHPLC system was equipped with C18 analytical column (50mm×2.1mm, 1.8μm), utilizing a mobile phase composed of pH 2.5 triethanolamine phosphate buffer (0.1M) and acetonitrile in a gradient elution mode; under these conditions six major catechins and caffeine were separated in a 3min run. The method was fully validated in terms of precision, detection and quantification limits, linearity, accuracy, and it was applied to the identification and quantification of catechins and caffeine present in green tea infusions. In particular, commercially available green tea leaves samples of different geographical origin (Sencha, Ceylon Green and Lung Ching) were used for infusion preparations (water at 85°C for 15min). The selectivity of the developed UHPLC method was confirmed by comparison with UHPLC-MS/MS analysis. The recovery of the main six catechins and caffeine on the three analyzed commercial tea samples ranged from 94 to 108% (n=3). Limits of detection (LOD) were comprised in the range 0.1-0.4μgmL(-1). An orthogonal micellar electrokinetic (MEKC) method was applied for comparative purposes on selectivity and quantitative data. The combined use of the results obtained by the two techniques allowed for a fast confirmation on quantitative characterization of commercial samples.

Histone post-translational modifications by HPLC-ESI-MS after HT29 cell treatment with histone deacetylase inhibitors

The goal of the present work is to establish a correlation between the degree of histone post‐translational modifications and the effects caused by treatment of HT29 colon cancer cells with class I‐selective (MS‐275 and MC1855), class II‐selective (MC1568), and non‐selective (suberoylanilide hydroxamic acid (SAHA) histone deacetylase inhibitors (HDACi). This correlation could afford a mean to better understand the mechanism of action of new, more potent, and selective HDACi directly on the cells. To this end, LC coupled to MS was applied in studies of time and concentration‐dependent treatment with HDACi in HT29 cells. The results were correlated to their potency of histone deacetylase inhibition and to their effects on the cell cycle. The results indicate that the four tested inhibitors show a different pattern of time‐ and concentration‐dependent modification after treatment of HT29 cells. At the selected concentrations, they cause different histone hyperacetylation and different cell cycle effects. In particular, SAHA (non‐selective HDACi) affected hyperacetylation of all histones and caused massive cell death. MC1855 (class I‐selective HDACi, hydroxamate) proved to be more potent and less toxic (cell arrest in G2/M phase) than SAHA. MS‐275 (class I‐selective HDACi, benzamide) exhibited a higher degree of hyperacetylation of H4 and a lower degree of H2A, H2B, and H3 acetylation, causing a cell arrest in G0/G1 phase. On the contrary, MC1568 (class II‐selective HDACi) produced only a modest hyperacetylation of H4, was ineffective on the other histones, and showed no effect on cell cycle in HT29 cells.

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases.

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.

A Fast and Validated Mass Spectrometry Method for the Evaluation of Human Serum Albumin Structural Modifications in the Clinical Field

A relatively fast analytical method for the identification and quantification of the post-transcriptional changes (PTCs) occurring in circulating human serum albumin (HSA) was developed. HSA is the most abundant protein in plasma and it represents the main determinant of plasma oncotic pressure, thus being the main modulator of fluid distribution between body compartments. Cirrhotic patients have low levels of HSA. Moreover, recent studies have demonstrated that during liver cirrhosis HSA presents PTCs affecting its properties. The HSA isoforms derived from these modifications could represent promising biomarkers for liver disease. Human plasma samples were collected from a cirrhotic patient (CH) and from an aged-matched non-cirrhotic subject (CT), purified by reverse-phase chromatography and analysed by an electrospray ionization quadrupole time-of-flight (ESI-Q-ToF) spectrometer. The deconvoluted ESI mass spectra from healthy subjects were all characterized by peaks attributed to mercaptoalbumin, nitrosylated, cysteinylated, glycated and N-terminal truncated HSA isoforms. The relative abundance of each isoform was derived and transformed into a relative per cent amount and the results were compared to those obtained analysing HSA from a CH plasma. The method was validated in terms of intra-day and inter-day reproducibility, both for quantitative results and PTCs molecular weight determination. The optimized method resulted in being effective in disclosing changes in HSA isoforms relative abundance and then it could be used for the systematic screening of cirrhotic patients to identify promising new biomarkers for liver diseases.

Towards automation in protein digestion: Development of a monolithic trypsin immobilized reactor for highly efficient on-line digestion and analysis

Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5mm×5.2mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90±9s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.

Ischaemia‐modified albumin: a marker of bacterial infection in hospitalized patients with cirrhosis

Patients with cirrhosis present structural changes of human serum albumin (HSA) affecting non‐oncotic functions. Ischaemia‐modified albumin (IMA), which reflects the capacity to bind cobalt, has been associated to patient mortality during acute‐on‐chronic liver failure. This study aimed to assess whether circulating IMA is elevated in advanced cirrhosis and its relationship with severity of cirrhosis and specific complications.

Disclosure of a fundamental clue for the elucidation of the myricetin mechanism of action as amyloid aggregation inhibitor by mass spectrometry.

Progression of Alzheimers disease involves aggregation of amyloid‐beta (Aβ) peptides, a complex process that involves the formation of several soluble intermediates and ends up with the deposition of ordered fibrillar architectures. The determination of the Aβ42 self‐assembly species targeted by an inhibitor is a key step in the identification of new inhibitors endowed with a suitable profile. In this context, the subtle characterization of myricetin (Myr) mode of action at a molecular level was performed by using different MS techniques, which allowed the monitoring of the modifications induced by Myr on the first occurring Aβ42 self‐assembly species. Results showed a persistent level of monomer and decreased formation of ordered Aβ42 aggregates in the presence of Myr, further, nano‐LC‐nano‐ESI‐QTOF, MALDI‐TOF, and ESI‐IT highlighted the formation of a new oxidized Aβ42 species, which is less prone to aggregation, in the presence of Myr. Coupling tryptic digestion and nano‐LC‐nano‐ESI‐QTOF also allowed the identification of Met35 as the specific site of oxidation along the Aβ aminoacid chain. Therefore, the detailed investigation by different MS techniques allowed better understanding of the molecular modification at the basis of the antiaggregating properties of Myr and highlighted its oxidizing action on the residue Met35 in Aβ monomers.

Mechanism and stereoselectivity of HDAC I inhibition by (R)-9-hydroxystearic acid in colon cancer.

9-Hydroxystearic acid (9-HSA) belongs to the endogenous lipid peroxidation by-products that decrease in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. It acts as a histone deacetylase (HDAC, E.C 3.5.1.98) inhibitor, and the interaction of the two enantiomers of 9-HSA with the catalytic site of the enzyme, investigated by using a molecular modelling approach, has been reported to be different. In this work we tested out this prediction by synthesizing the two enantiomers (R)-9-HSA (R-9) and (S)-9-HSA (S-9) starting from the natural source methyl dimorphecolate obtained from Dimorphotheca sinuata seeds and investigating their biological activity in HT29 cells. Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, R-9 being more active; R-9 and S-9 inhibitory effect induces an increase in histone H4 acetylation. We also demonstrate that the antiproliferative effect brought about by R-9 is more pronounced as well as we observe increase of p21 transcription and protein content, while the expression of cyclin D1 is decreased. Starting from these observations it can be hypothesized that the interaction of R-9 with HDAC1 induce conformational changes in the enzyme causing loss of its interaction with other proteins, like cyclin D1 itself.