Cosima Damiana Calvano

Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry

Protein glycosylation is a common post-translational modification that is involved in many biological processes, including cell adhesion, protein-protein and receptor-ligand interactions. The glycoproteome constitutes a source for identification of disease biomarkers since altered protein glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin-based and HILIC-based affinity enrichment techniques, alone or in combination, for preparation of glycoproteins and glycopeptides for subsequent analysis by MALDI and ESI mass spectrometry. The methods were successfully applied to human serum samples and a total of 86 N-glycosylation sites in 45 proteins were identified using a mixture of three immobilized lectins for consecutive glycoprotein enrichment and glycopeptide enrichment. The combination of lectin affinity enrichment of glycoproteins and subsequent HILIC enrichment of tryptic glycopeptides identified 81 N-glycosylation sites in 44 proteins. A total of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer patients and healthy individuals to assess glycosylation site frequencies.

MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent

The adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HO) is frequent and constitutes a serious concern both for oil suppliers and consumers. The high degree of similarity between the two oils as regards triacylglycerol, total sterol and fatty acid profile, complicates the detection of low percentages of HO in EVOO. However, phospholipids (PLs) are usually present in seed oils at a concentration range of 10-20 g/kg, while the amounts of PLs in VOOs are 300-400 times lower. Thus, in this work a sample pretreatment procedure focused towards the selective PLs extraction was developed; the Bligh-Dyer extraction procedure was modified introducing the ionic liquid resulting from the combination of TBA (tributylamine) and CHCA (α-Cyano-4-hydroxycinnamic acid) as extraction solvent. The selective extraction and enrichment of phospholipids from EVOO and HO samples was then achieved. The relevant extracts were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using the same ionic liquid TBA-CHCA as MALDI matrix, that was found to be very suitable for PLs analysis. In fact, a remarkable increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, was observed in the relevant mass spectra. The applicability of the whole method to the individuation of the presence of HO in EVOO was demonstrated by the analysis of EVOO samples progressively adulterated with variable quantities of HO, that was still detectable at a 1% contamination level.

Determination of clenbuterol in human urine and serum by solid-phase microextraction coupled to liquid chromatography.

A solid-phase microextraction (SPME)-LC-UV method for the determination of the beta-adrenergic drug clenbuterol in human urine and serum samples was developed for the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The procedure required very simple sample pretreatments, isocratic elution, and provided highly selective extractions. All the aspects influencing fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte have been investigated. The linear ranges investigated in urine and serum were 10-500 and 5-500 ng/ml, respectively (that covers the typical clenbuterol concentration observed in biological fluids). Within-day and between-days R.S.D.% in urine ranged between 5.0-5.3 and 8.5-8.7, respectively, while in serum ranged between 5.5-5.9 and 8.7-9.1, respectively. Estimated LOD and LOQ were 9 and 32 ng/ml (spiked urine), respectively, and 5 and 24 ng/ml (spiked serum), respectively, well below the usual clenbuterol urinary and serum level.

Aniline/α-cyano-4-hydroxycinnamic acid is a highly versatile ionic liquid for matrix-assisted laser desorption/ionization mass spectrometry

The performance of a matrix-assisted laser desorption/ionization (MALDI) ionic liquid matrix (ILM) consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA) and aniline (ANI) was evaluated to assess whether it could offer possible advantages over conventional matrices. Ultraviolet (UV), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and laser desorption/ionization mass spectrometry (LDI-MS) experiments were carried out with the aim of confirming the structure of the ANI-CHCA ILM. Different model analytes such as amino acids, peptides, proteins, lipids, phospholipids, synthetic polymers, and sugars were tested. Mass spectra with similar or improved signal-to-noise (S/N) ratio (compared to CHCA) were invariably obtained demonstrating the potential of this ILM as a general purpose matrix. Furthermore, protein identification by peptide mass fingerprinting (PMF) and database search was facilitated compared to CHCA since higher scores and increased sequence coverage were observed. Finally, a complex lipid mixture (i.e. a raw extract of a milk sample) analysed by MALDI-MS showed improved S/N ratio, a reduced chemical noise and a limited formation of matrix-clusters.

Proteomic Approach Based on MALDI-TOF MS To Detect Powdered Milk in Fresh Cow’s Milk

Milk and cheese are expensive foodstuffs, and their consumption is spread among the population because of their high nutritional value; for this reason they are often subjected to adulterations. Among the common illegal practices, the addition of powdered derivatives seems very difficult to detect because the adulterant materials have almost the same chemical composition of liquid milk. However, the high temperatures (180-200 °C) used for milk powder production could imply the occurrence of some protein modifications (e.g., glycation, lactosylation, oxidation, deamidation, dehydration). The modified proteins or peptides could then be used as markers for the presence of powdered milk. In this work, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze tryptic digests relevant to samples of raw liquid (without heat treatment), commercial liquid, and powdered cows milk. Samples were subjected to two-dimensional gel electrophoresis (2-DE); differences among liquid and powder milk were detected at this stage and eventually confirmed by MALDI analysis of the in gel digested proteins. Some diagnostic peptides of powdered milk, attributed to modified whey proteins and/or caseins, were identified. Then, a faster procedure was optimized, consisting of the separation of caseins from milk whey and the subsequent in-solution digestion of the two fractions, with the advantage of obtaining almost the same information in a limited amount of time. Finally, analyses were carried out with the fast procedure on liquid milk samples adulterated with powdered milk at different percentages, and diagnostic peptides were detected down to 1% of adulteration level.

Selective extraction of phospholipids from dairy products by micro-solid phase extraction based on titanium dioxide microcolumns followed by MALDI-TOF-MS analysis

A new micro-solid phase extraction (µ-SPE) procedure based on titanium dioxide microcolumns was developed for the selective extraction of phospholipids (PLs) from dairy products before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. All the extraction steps (loading, washing, and elution) have been optimized using a synthetic mixture of PLs standard and the procedure was subsequently applied to food samples such as milk, chocolate milk and butter. The whole method demonstrated to be simpler than traditional approaches and it appears very promising for a rapid PLs screening and characterization also in biological matrices.

Lipid fingerprinting of Gram-positive lactobacilli by intact cells – matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge based matrix

A method of direct lipid analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in intact membranes, without prior extraction/separation steps, is described. Here, we demonstrate the efficacy of a strong base, 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-time-of-flight (TOF) MS analysis of whole cell bacteria. Initially, individual acidic low-molecular-weight analytes such as standard free fatty acids and phospholipids were analyzed using DMAN as matrix. Clear negative-mode MALDI-TOF MS spectra of all analytes show only deprotonated analyte signals at a low picomole limit of detection with the complete absence of matrix-related signals. These results indicate that DMAN represents a suitable matrix for MALDI-TOF MS analysis of mixtures of complex lipids as the intact membranes of microorganisms. DMAN was successfully applied to the analysis of Lactobacillus sanfranciscensis and L. plantarum microorganisms. Different components were sensitively detected in a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids, glycolipids, phosphatidylglycerols (PGs) and cardiolipins. This method might be of general application, offering the advantage of quickly gaining information about lipid components of other gram-positive bacterial membranes.

A simple protocol for Matrix Assisted Laser Desorption Ionization- time of flight-mass spectrometry (MALDI-TOF-MS) analysis of lipids and proteins in single microsamples of paintings

A simple protocol, based on Bligh-Dyer (BD) extraction followed by MALDI-TOF-MS analysis, for fast identification of paint binders in single microsamples is proposed. For the first time it is demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. The protocol makes use of an alternative denaturing anionic detergent (RapiGest™) in order to improve efficiency of protein digestion and purification step. Detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of pigments on ageing of lipids and proteins was also investigated. Finally, the proposed protocol was successfully applied to the study of a late-15th century Italian panel painting allowing the identification of various proteinaceous and lipid sections in organic binders, such as egg yolk, egg white, animal glue, casein, and drying oil.

MALDI-TOF MS Characterization of Glycation Products of Whey Proteins in a Glucose/Galactose Model System and Lactose-free Milk

The major modifications induced by thermal treatment of whey proteins α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) in a model system mimicking lactose-free milk (L(-) sugar mix) were investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The analysis of the intact α-La revealed species with up to 7 and 14 adducts from lactose and sugar mix, respectively, whereas for β-Lg 3 and up to 5 sugar moieties were observed in the case of lactose and sugar mix experiments, respectively. A partial enzymatic hydrolysis with endoproteinase AspN prior to mass spectrometric analysis allowed the detection of further modifications and their localization in the amino acid sequence. Using α-cyano-4-chlorocinnamic acid as MALDI matrix, it could be shown that heating α-La and β-Lg with glucose or galactose led to the modification of lysine residues that are not glycated by lactose. The higher glycation degree of whey proteins in a lactose-free milk system relative to normal milk with lactose reflects the higher reactivity of monosaccharides compared to the parent disaccharide. Finally, the analysis of the whey extract of a commercial lactose-free milk sample revealed that the two whey proteins were present as three main forms (native, single, and double hexose adducts).

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor

Significance We have shown previously that oxytocin (Oxt), other than regulating lactation and social bonding, is a potent stimulator of bone formation by the osteoblast. Here, we present evidence that this action is exerted through the nuclear localization of the Oxt receptor (Oxtr). Our findings prompt additional studies into the contribution of nuclear Oxtr signaling in regulating lactation and social bonding. We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr–EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.