Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression.

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.

A widespread picture of the Streptococcus thermophilus proteome by cell lysate fractionation and gel-based/gel-free approaches.

Among the group of lactic acid bacteria, Streptococcus thermophilus has found a wide application in industrial processes used for the manufacture of dairy products. Taking advantage of different proteome extraction and subfractionation protocols, bacterial cytosolic and membrane proteins were isolated and resolved by independent gel-free and gel-based separation procedures. Whole cytosolic fraction and its acid, basic and low molecular mass protein components were separated by different resolutive 2-DE and tricine 1-DE gels and identified by MALDI-TOF PMF and/or microLC-ESI-IT-MS/MS. Membrane proteins were resolved by 2-DE and SDS-PAGE gels and similarly identified by PMF and TMS analysis. In parallel, whole extract was trypsinized and resulting peptides were identified by shotgun 2-D LC-ESI-IT-MS/MS analysis. Using this combined approach, expression products corresponding to 458 different genes were identified, which cover almost a third of the predicted vegetative proteome. Relative protein concentration and hydrophobicity affected protein detection. Broad recognition was obtained for enzymes involved in carbohydrate, fatty acid, amino acid and nucleotide metabolism, replication, transcription, translation, cell wall synthesis, as well as for proteins affecting bacterial functions important for industrial applications, i.e. milk sugar import and exopolysaccharide biosynthesis. By providing detailed reference electrophoretic/chromatographic maps to be used in future comparative proteomic investigations on bacteria grown under various experimental conditions or on different bacterial strains, our results will favour dedicated studies on S. thermophilus metabolism and its regulation or on detection of biomarkers for selection of optimal strains for industrial applications.

A study of Streptococcus thermophilus proteome by integrated analytical procedures and differential expression investigations.

Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.

Comparative proteomic analysis of mammalian animal tissues and body fluids: bovine proteome database.

Characterizing the complete proteome of multicellular organisms is a challenging task using the currently available technologies. With the increasing degree of genetic complexity, animals acquire a broader repertoire of options to meet environmental challenges. Mammalian cells from different tissues/body fluids express different thousands of proteins with a predicted dynamic range of up to five to six orders of magnitude, thus necessitating the whole arsenal of dedicated analytical strategies for a detailed proteome characterization. Nevertheless, 2D-E analysis of whole cellular lysates still remains the most used initial approach for the proteomic description of specialized cells. It enables to obtain an overview of the main soluble protein components of a specific tissue/body fluid, allowing comparison between different cellular types and molecular description of organ specialization. Massive proteomic investigations have been reported mainly in the case of human, mouse and rat, allowing comparative analysis. For this reason, a research project focused on the 2D-E characterization of tissues and biological fluids from other domestic mammals has been undertaken in our laboratory. A number of high-resolution reference electrophoretic maps have been established for liver, kidney, muscle, plasma and red blood cells samples from Holstein Friesian bovine female individuals. Among the 1863 distinct protein features detected, 534 species were identified and associated to 209 different genes by a combination of MALDI-TOF mass fingerprint, capillary LC-ESI-IT-MS-MS and image gel matching procedures. Identified polypeptide species and differences in expression profiles between various tissues/fluids clearly reflected organ biochemical specialization. This experimental output allowed establishing a 2D-E bovine database accessible at the URL address for image comparison.

Differential proteomic analysis in the study of prokaryotes stress resistance.

Proteomic technologies are powerful tools to study the physiological response of bacteria to various environmental stress conditions. Complex protein toolkits coordinated by regulatory networks have evolved to accommodate bacterial survival under several stressing conditions, such as varying temperature or pH, and changes in nutrient availability. This manuscript provides an overview of the major achievements obtained by 2D gel based and non-gel based proteomic approaches for the study of adaptive response in bacteria. As an example, a study carried out on Streptococcus thermophilus response to various stresses is here reported. In this investigation, we used combined proteomic strategies to define proteomic maps useful to highlight protein profile variations following environmental challenges.

Mechanistic studies on bovine cytosolic 5'-nucleotidase II, an enzyme belonging to the HAD superfamily.

Cytosolic 5'-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho-enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5'-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.

Probing the dimeric structure of porcine aminoacylase 1 by mass spectrometric and modeling procedures.

Aminoacylase 1 is a zinc-binding metalloprotease catalyzing the hydrolysis of N(alpha)-acylated l-amino acids; it presents altered expression levels in different renal and small cell lung carcinomas. A description of its redox and oligomerization state was achieved by combined biochemical and mass spectrometric procedures. A topological analysis of the enzyme structural architecture was derived from limited proteolysis and selective chemical modification experiments, using a broad range of proteases and chemical reagents. The analysis of the reaction products by different mass spectrometric techniques identified 26 amino acids as being accessible on the molecular surface, defining polypeptide regions exposed in the structure of the dimeric protein. The nature of the intermolecular contact zone between monomers was investigated by cross-linking reaction and mass mapping experiments. The cross-linked dimer was isolated, and the intermolecular cross-linked peptides were characterized, thus demonstrating the spatial proximity of Lys220 and Lys231 at the dimerization interface. Standard modeling procedures based on automatic alignment on the structure of members of the M20 peptidase family failed to produce a dimeric model consistent with experimental data. Discrepancies were observed mainly at the dimer interface and at loop regions. Therefore, a refined model for this dimeric protease was calculated by selecting the one able to generate a structure fully compatible with experimental findings, among all possible suboptimal sequence alignments. According to this model, each aminoacylase monomer consists of two domains: a globular catalytic subunit (residues 1-188 and 311-399) consisting of a beta-sheet sandwiched between alpha-helices and a second beta-sheet located on the surface, and the dimerization domain (residues 189-310) folding into a beta-sheet flanked on one side by two alpha-helices. These results indicate that reliable approaches such as limited proteolysis, selective chemical modification, and cross-linking coupled to mass spectrometry can be used to test and optimize molecular models of multimeric proteins and highlight problems in automatic model building.

Proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver, kidney, muscle, plasma and red blood cells.

A number of high resolution two-dimensional electrophoresis (2-DE) reference maps for bovine tissues and biological fluids have been determined for animals in basal state. Among the 1863 distinct protein features detected in samples of liver, kidney, muscle, plasma and red blood cells, 509 species were identified and associated to 209 different genes. Difficulties in the identification were related to the poorly characterized Bos taurus genome and were solved by a combined matrix-assisted laser desorption/ionisation-mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry approach. The experimental output allowed us to establish a 2-DE database accessible through the World Wide Web network at the URL address (http://www.iabbam.na.cnr.it/Biochem). These reference maps may serve as a tool in future veterinary medical studies aimed at the evaluation of changes in protein repertoire for altered animal physiological conditions and infectious diseases, to the definition of molecular markers for novel diagnostic kits and vaccines, as well as the characterization of protein modifications in bovine materials following technological processes used in the food industry.

Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma.

Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

Characterization of heat-induced lactosylation products in caseins by immunoenzymatic and mass spectrometric methodologies.

Nonenzymatic glycosylation of proteins occur during milk thermal treatment through the Maillard reaction. Immunoenzymatic and spectrometric methodologies were used to investigate caseins modification in samples submitted to different processing. As expected, protein-bound carbonyl content in raw and thermal-treated milks was positively correlated with the severity of the treatment. When immunoblotting and ELISA experiments were carried out either on isolated or whole milk proteins, carbonyl accumulation on caseins and macromolecular aggregates produced by thermal processing was evidenced. A comparative electrospray-mass spectrometry (ES-MS) analysis of different milks allowed verifying the extent of Amadori adducts on the more abundant protein components (alphas1- and beta-casein (alphas1- and beta-CN)). A combined matrix-assisted laser desorption ionization (MALDI)-MS/Edman degradation approach on enzymatic digests identified caseins modification sites. In moderately heat-treated milks, we observed that lactosylation occurred specifically at Lys-34 (alphas1-CN) and Lys-107 (beta-CN); whereas, samples subjected to more severe conditions presented modification at Lys-7, Lys-34, Lys-83, Lys-103, Lys-105, Lys-132 and Lys-193 (alphas1-CN) and at Lys-32, Lys-48, Lys-107, Lys-113 and Lys-176 (beta-CN). Differences in secondary structure of these components was assayed by circular dichroism (CD) spectroscopy.