Rosaria Saletti

Proteome analysis of Citrus sinensis L. (Osbeck) flesh at ripening time

A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.

Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

Poppea's bath liquor: The secret proteome of she-donkey's milk

Donkeys milk is today categorized among the best mothers milk substitute for allergic newborns, due to its much reduced or absent allergenicity, coupled to excellent palatability and nutritional value. However, up to the present, only a handful of proteins had been characterized, just about the standard eight to ten major ones known in all types of milk. By exploiting the combinatorial peptide ligand library technology, and treating large volumes (up to 300 mL) of defatted, de-caseinized (whey) milk, we have been able to identify 106 unique gene products, by far the largest description so far of this precious nutrient. Due to poor knowledge of the donkeys genetic asset, only 10% of the proteins could be identified by consulting the data base of Equus asinus; the largest proportion (70%) could be identified by homology with the proteins of Equus caballus.

Mass spectrometry in food proteomics: a tutorial †

In the last decades, the continuous and rapid evolution of proteomic approaches has provided an efficient platform for the characterization of food-derived proteins. Particularly, the impressive increasing in performance and versatility of the MS instrumentation has contributed to the development of new analytical strategies for proteins, evidencing how MS arguably represents an indispensable tool in food proteomics. Investigation of protein composition in foodstuffs is helpful for understanding the relationship between the protein content and the nutritional and technological properties of foods, the production of methods for food traceability, the assessment of food quality and safety, including the detection of allergens and microbial contaminants in foods, or even the characterization of genetically modified products. Given the high variety of the food-derived proteins and considering their differences in chemical and physical properties, a single proteomic strategy for all purposes does not exist. Rather, proteomic approaches need to be adapted to each analytical problem, and development of new strategies is necessary in order to obtain always the best results. In this tutorial, the most relevant aspects of MS-based methodologies in food proteomics will be examined, and their advantages and drawbacks will be discussed.

Applications of mass spectrometry techniques in the investigation of milk proteome

The introduction of “soft” desorption/ionization methods such as electrospray ionization and matrix-assisted laser desorption/ionization has determined a breakthrough in the application of mass spectrometry to the structural analysis of proteins. The contemporary advancement of bioinformatics, together with the possibility to combine these mass spectrometric methods with electrophoretic or chromatographic separation techniques has opened up the new field of proteome analysis and, more generally, has established these approaches as indispensable tools for protein and peptide analysis in complex mixtures, such as milk and milk-derived foods. Here, a necessarily not exhaustive series of current applications of mass spectrometry-based techniques for the characterization of milk proteins will be summarized. These include the characterization of milk protein polymorphism, determination of the structural modifications induced on milk proteins by industrial processes, investigation of milk adulterations and characterization of milk allergens.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

Sequence determination of αs1-casein isoforms from donkey by mass spectrometric methods

Four co-eluting components, with experimentally measured M(r) of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions, MALDI-TOF MS and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analyses, the four components were identified as donkeys alpha(s1)-CNs and their sequences completely characterized, using the known mares alpha(s1)-CN (GenBank Acc. No. AAK83668; M(r) 23750.7 Da) as reference. The proteins with M(r) of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full-length component and present the amino acid substitutions Q(8)-->H and H(115)-->Y with respect to the mares alpha(s1)-CN. The other two components with M(r) 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full-length component, show the insertion of the pentapeptide HTPRE between Leu(33) and the Glu(34). The two alpha(s1)-CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M(r) 24 406) and A(1) (201 amino acids; M(r) 24 278), whereas the two alpha(s1)-CNs without the pentapeptide were named variants B (197 amino acids; M(r) 23 786) and B(1) (196 amino acids; M(r) 23 658).

Sequence and phosphorylation level determination of two donkey β‐caseins by mass spectrometry

Two coeluting components, with experimentally measured M(r) values of 25529 and 24606 Da, were identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis, the two proteins were identified as donkey beta-CNs and their sequences characterized completely, using the two known beta-CNs from mare as references. The two donkey beta-CNs, showing a mass difference of 923 Da, differ by the presence of the domain E(27)SITHINK(34) in the full-length component (M(r) 25529 Da). In comparison with the mares beta-CNs used as reference, they present nine amino acid substitutions: L-->S(37), R-->H(52), S-->N(81), P-->V(84), L-->V(91), R-->Q(203), P-->L/I(206), L-->F(210) and A-->P(219). Together, these substitutions account for the increase of 18 Da in the M(r) of the donkey beta-CNs with respect to the counterparts from the mare. The molecular mass determination by ESI-MS for the phosphorylated proteins showed that the full-length component was composed of highly multi-phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mares beta-CNs, the full-length (226 amino acids) beta-CN was termed variant A, whereas the shorter (218 amino acids) beta-CN was termed variant A(Delta5).

MALDI-TOF mass spectrometry for the monitoring of she-donkey's milk contamination or adulteration

Donkeys milk (DM), representing a safe and alternative food in both IgE-mediated and non-IgE-mediated cows milk protein allergy, can be categorized as precious pharma-food. Moreover, an economically relevant interest for the use of DM in cosmetology is also developing. The detection of adulterations and contaminations of DM is a matter of fundamental importance from both an economic and allergenic standpoint, and, to this aim, fast and efficient analytical approaches to assess the authenticity of this precious nutrient are desirable. Here, a rapid matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based method aimed to the detection of bovine or caprine milk in raw DM is reported. The presence of the extraneous milks was revealed by monitoring the protein profiles of the most abundant whey proteins, α-lactalbumin (α-LA) and β-lactoglobulin, used as molecular markers. The possibility of obtaining a quantitative analysis of the level of cow or goat milk in DM based on the MALDI-TOF peak areas of α-LAs was also explored. The results showed that the experimental quantitative values were in good agreement with the real composition of each mixture. As pretreatment of the milk samples is not required, and owing to the speed and the high sensitivity of MALDI-MS, the protocol here reported could represent a reliable method for routine analyses aimed to assess the absence of contamination in raw fresh DM samples.