Milena Quaglia

Surface initiated molecularly imprinted polymer films: a new approach in chiral capillary electrochromatography

A new generation of imprinted composite particles was tested as capillary electrochromatography stationary phase. Silica particles characterised by a well defined particle size (10 µm diameter), shape and pore system (1000 A) were modified with an azoinitiator and subsequently used to graft molecularly imprinted polymers targeted to bind L-phenylalanine anilide. Fused silica capillaries were packed over a length corresponding to 8 cm, using a pneumatic amplification pump, and the stationary phase thus obtained was tested with respect to its electrochromatographic performance. The electroendosmotic flow mobility was evaluated with respect to both the different content of polymer on the silica particle surface and different operating pH values. The dependence of various parameters, namely the analyte concentration, the polymer layer thickness and the pH of the mobile phase on the enantioselectivity was investigated. These CEC capillaries showed enantioselectivity comparable with that showed in LC, and exhibited improved performance in terms of plate number N1 (∼13 000), selectivity α (∼1.5), analysis time (<3 min), inter–intraday and intercapillary reproducibility. We expect this approach to result in a new generation of robust, tailor-made chiral or affinity stationary phases for CEC.

An enantioselective imprinted receptor for Z-glutamate exhibiting a binding induced color change

Using 1-(4-styryl)-3-(3-nitrophenyl)urea as host monomer for the imprinting of Z-(D or L)-Glu, a polymeric receptor exhibiting strong enantioselectivity and a change in color intensity upon binding of the guest was obtained.

Quantification of Human Growth Hormone in Serum with a Labeled Protein as an Internal Standard: Essential Considerations

To manage and inform diagnostic or therapeutic decisions, measurement results which are accurate, specific, and comparable between laboratories are required. Two challenges associated with this are the definition of the measurand and the commutability of the reference standard used. Once the measurand is defined, the next step in improving standardization is developing traceable quantification methods for proteins in biological fluids. A novel reference method for the quantification of recombinant human growth hormone (rhGH) in serum has been developed using multistep sample cleanup at the protein level, tryptic digestion, and isotope dilution mass spectrometry (IDMS). Critical considerations for using isotopically labeled rhGH as the internal standard are described. A bulk serum sample was prepared at the clinically relevant level of 10 ng/g and quantified using the method described to give results traceable to the International System of Units (SI) with a total measurement uncertainty of <20%. Results compared favorably with an orthogonal traceable method using total tryptic digestion, peptide separation, and isotope dilution mass spectrometry.

Sulfonated molecules that bind a partially structured species of β2‐microglobulin also influence refolding and fibrillogenesis

Human β2‐microglobulin (β2‐m) is a small amyloidogenic protein responsible for dialysis‐related amyloidosis, which represents a severe complication of long‐term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of β2‐m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of β2‐m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.

Evaluation of quail egg white riboflavin binding protein as a chiral selector in capillary electrophoresis by applying a modified partial filling technique

A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on‒line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol‒coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 μM up to 900 μM and different plug lengths, while the running buffer pH (6.0), temperature (25oC) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine.

Capillary electrophoresis in drug discovery.

The several advantages that capillary electrophoresis (CE) offers in the study of protein folding, protein-ligand and protein-protein interactions, render this methodology appealing in several areas. In this chapter, a specific example is reported, where the use of affinity CE (ACE) in drug discovery is particularly advantageous over other separative and spectroscopic techniques. ACE is an analytical approach in which the migration patterns of interacting molecules in an electric field are recorded and used to identify specific binding and to estimate binding constants. A library of compounds has been tested, in free solution and with minimum sample consumption, for the affinity to two targets previously separated by CE, the native form and the partially structured intermediate of the folding of beta(2)-microglobulin (beta(2)-m) [Chiti et al. (J. Biol. Chem. 276:46714-46721, 2001), Quaglia et al. (Electrophoresis 26:4055-4063, 2005)]. beta(2)-m is an intrinsically amyloidogenic protein, and its tendency to misfold is responsible for dialysis-related amyloidosis, an unavoidable complication of chronic haemodialysed patients. The criteria for choosing the compounds to be screened, the method conditions, and the possible data analysis strategies are detailed and discussed in this chapter.

Assessment of Recovery of Milk Protein Allergens from Processed Food for Mass Spectrometry Quantification

Assessing the recovery of food allergens from solid processed matrixes is one of the most difficult steps that needs to be overcome to enable the accurate quantification of protein allergens by immunoassay and MS. A feasibility study is described herein applying International System of Units (SI)-traceably quantified milk protein solutions to assess recovery by an improved extraction method. Untargeted MS analysis suggests that this novel extraction method can be further developed to provide high recoveries for a broad range of food allergens. A solution of α-casein was traceably quantified to the SI for the content of α-S1 casein. Cookie dough was prepared by spiking a known amount of the SI-traceable quantified solution into a mixture of flour, sugar, and soya spread, followed by baking. A novel method for the extraction of protein food allergens from solid matrixes based on proteolytic digestion was developed, and its performance was compared with the performance of methods reported in the literature.

Development of a LC-MS method for the discrimination between trace level Prunus contaminants of spices

The need for an analytical procedure for the identification of allergens present at trace levels in foods was highlighted by conflicting results in a case of contamination of the spice cumin. The application of a bottom-up proteomics experiment was investigated to identify marker peptides for potential contaminant nuts which could then be monitored with high specificity and sensitivity by selective reaction monitoring experiments. The method developed allowed for the distinction between two closely related Prunus species, almond and mahaleb, in two different spices, cumin and paprika. The paprika sample was found to be contaminated with almond and the cumin sample, contaminated at a much lower level, was found to be contaminated with mahaleb. The method could be applied to any protein-dense food matrix allergen so long as suitable control and reference samples can be acquired.

Almond or Mahaleb? Orthogonal Allergen Analysis During a Live Incident Investigation by ELISA, Molecular Biology, and Protein Mass Spectrometry

It is now well known that an incident investigated in the United Kingdom in 2015 of cumin alleged to be contaminated with almond, a risk for people with almond allergy, was caused by the Prunus species, Prunus mahaleb. In the United Kingdom, the Government Chemist offers a route of technical appeal from official findings in the food control system. Findings of almond in two official samples, cumin and paprika, which had prompted action to exclude the consignments from the food chain, were so referred. Herein are described the approaches deployed to resolve the analytical issues during the investigation of the incidents. The cross-reactivity of ELISA to Prunus species was confirmed, and although this is useful in screening for the genus, orthogonal techniques are required to identify the species and confirm its presence. Two novel PCR assays were developed: one specific for P. mahaleb and the other a screening method capable of identifying common Prunus DNA. Peptides unique to almond and mahaleb were identified, permitting LC-tandem MS and criteria were developed for peptide identification to forensic standards. This work enables a staged approach to be taken to any future incident thought to involve Prunus species and provides a template for the investigation of similar incidents.

BETTER MEASUREMENT FOR IMPROVED DIAGNOSIS AND MANAGEMENT OF ALZHEIMER'S DISEASE: UPDATE ON THE EMPIR NEUROMET PROJECT

The development of novel therapies for Alzheimer’s Disease (AD) is constrained by the lack of available methods for preclinical diagnosis, despite extensive research on biomarker identification. He ...