Grasping the proteome: detergents sub-fractionation of human and microbial cells leads to improvements in differential proteomics

La mia tesi di dottorato approfondisce aspetti relativi all\u2019analisi proteomica in gel bidimensionale: il lavoro svolto si e\u2019 proposto di apportare da un lato un contributo al miglioramento dei metodi di pre-frazionamento del campione, finalizzato al raggiungimento di un alto livello di risoluzione, ad una selezione di popolazioni di proteine pi\uf9 omogenee per la separazione su gel, e ad una migliore riproducibilit\ue0 delle mappe proteiche, e dall\u2019altro l\u2019impiego dei protocolli messi a punto nella prima fase della ricerca e\u2019 stato immediatamente trasferito e messo alla prova su due problematiche specifiche e selezionate di ambito sia microbiologico, che biomedico. L\u2019applicazione dei protocolli di pre-frazionamento unitamente ad un disegno sperimentale di proteomica differenziale ha permesso di ricavare importanti informazioni biochimico-cellulari e di operare correlazioni stimolo-effetto. Gli obiettivi della tesi sono stati: 1) messa a punto di protocolli efficaci per la eliminazione di contaminanti (acidi nucleici e polisaccaridi) dal campione proteico per 2-DE; 2) indagine proteomica differenziale per delucidare i meccanismi molecolari dell\u2019acido-resistenza di Ga. hansenii AAB248; 3) messa a punto di protocolli di frazionamento del campione in: proteine di membrana, del citosol e proteine associate alle membrane; 4) indagine sull\u2019omeostasi del ferro nei macrofagi con approccio di proteomica differenziale: effetti della stimolazione con ferro ionico; 5) indagine sull\u2019omeostasi del ferro nei macrofagi con approccio di proteomica differenziale: effetti della stimolazione con eritrociti senescenti. Proteomica microbiologica In collaborazione con il Dott. Giacomo Zapparoli, laboratorio di microbiologia, Dipartimento di Biotecnologie, Universit\ue0 di Verona e con il Prof. Paolo Giudici, Dipartimento di Scienze Agrarie, Universit\ue0 degli Studi di Modena e Reggio Emilia. Proteomica biomedica In collaborazione con la Dott.ssa Annalisa Castagna e il Dott. Domenico Girelli, Dipartimento di Medicina Clinica e Sperimentale, Unit\ue0 di Medicina Interna B, Universit\ue0 di Verona, con la Dott.ssa Ivana De Domenico, Division of Hematology, Department of Medicine, School of Medicine, University of Utah, ed il Prof. Jerry Kaplan, Department of Pathology, School of Medicine, University of Utah.Proteomics is an emerging area of science that attempts to study proteins on a massively parallel scale. It is by essence a multidisciplinary science: physics, chemistry, bioinformatics and mathematics join biochemistry, biology and medicine to solve general life science questions. Scientists worldwide are applying proteomic technology to solve problems which cannot be resolved by traditional methods, particularly in the biochemical field. Methodologically, proteomics is based on highly efficient methods of separation and analysis of proteins in living systems, which need to be continuously improved in order to achieve and maintain high resolution standards. Proteomics is considered a key technology in many biomedical sectors such as molecular medicine, drug discovery, clinical diagnostics, as well as microorganisms and plant studies. Shared problem in proteomic analysis is the great complexity of the samples, thus the proteomic technology needs improvements and new contributes to overcome the actual limitations in the samples treatment and recovery, to shorten and ease the recovery protocols, to affirm reproducibility and to effectively remove contaminants, in order to reach the degree of automation and cross-laboratory reproducibility collectively expected by the scientific community. The objectives of this thesis were both the development of protocols for protein recovery in order to achieve higher resolution and reproducibility in 2-DE, and at the direct applications of the set methods to the understanding of two selected and interesting proteomic cases. Concerning microbiological proteomics, we studied the molecular mechanism of acid-resistance in Ga. Hansenii; and concerning the biomedical proteomics, the molecular basis of iron homeostasis in macrophages was studied. The thesis work was performed at the Biochemical Methodology and Proteomics laboratory of Department of Biotechnology of the University of Verona, in collaboration with other laboratories. Regarding the study on macrophages, we collaborated with Dr. Annalisa Castagna and Prof. Domenico Girelli, Unit of Internal Medicine B, Department of Clinical and Experimental Medicine of the University of Verona, with Dr. Ivana De Domenico, Division of Hematology, Department of Medicine, School of Medicine, University of Utah, and Prof. Jerry Kaplan, Department of Pathology, School of Medicine, University of Utah, and with Dr. Anna Maria Timperio and Prof. Lello Zolla, Department of Ambiental Science, University of \u201cLa Tuscia\u201d, Viterbo. Regarding the study on Ga. hansenii we collaborated with Dr. Giacomo Zapparoli, Department of Biotechnology of the University of Verona and with Prof. Paolo Giudici, Department of Agricultural and Food Science, University of Modena and Reggio Emilia

High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics

<p>Abstract</p> <p>Background</p> <p>Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels.</p> <p>Results</p> <p>Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis) indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm) we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS).</p> <p>Conclusion</p> <p>This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.</p

Polar Electrophoresis: Shape of Two-Dimensional Maps Is as Important as Size

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of “isobaric” spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis

A CTAB based method for the preparation of total protein extract of wine spoilage microrganisms for proteomic analysis.

Mapping the proteome of microrganisms by 2D-electrophoresis is often a hard task, because many contaminants, e.g. polysaccharides of the cell wall and nucleic acid, can obstruct the pores of the IEF gel resulting in streaks and smears. A protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed. The cellulose-producing strain Gluconoacetobacter hansenii AAB0248 was resolved on 7cm Minigels in over 500 protein spots (a hundred more than with protocols reported in literature). The method was further employed for mapping the proteome of some acid adapted, wine spoilage microrganisms e.g. acetic acid bacteria and a yeast

Improvements to polar 2-D electrophoresis for proteomic applications.

Recently, we reported a new way of performing 2-DE, called P-dimensional electrophoresis (2-PE). In this approach, the second dimension is achieved in a radial gel which can accommodate up to six 7 cm long IPG strips simultaneously, improving reproducibility and throughput power in respect to 2-DE. Nevertheless, 2-PE was up to now limited to the use of only short strips because of technical difficulties. Here, we describe how to load longer strips (e.g., 18-24 cm) on 2-PE and report some representative images for a qualitative assessment

Pros and cons of peptide isolectric focusing in shotgun proteomics

In shotgun proteomics, protein mixtures are proteolytically digested before tandem mass spectrometry (MS/MS) analysis. Biological samples are generally characterized by a very high complexity, therefore a step of peptides fractionation before the MS analysis is essential. This passage reduces the sample complexity and increases its compatibility with the sampling performance of the instrument. Among all the existing approaches for peptide fractionation, isoelectric focusing has several peculiarities that are theoretically known but practically rarely exploited by the proteomics community. The main aim of this review is to draw the readers' attention to these unique qualities, which are not accessible with other common approaches, and that represent important tools to increase confidence in the identification of proteins and some post-translational modifications. The general characteristics of different methods to perform peptide isoelectric focusing with natural and artificial pH gradients, the existing instrumentation, and the informatics tools available for isoelectric point calculation are also critically described. Finally, we give some general conclusions on this strategy, underlying its principal limitations

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged aging

The biological mechanisms involved in structural and biochemical changes during aging that are responsible of tenderization of meat are not yet completely understood. To study proteomic changes involved in tenderization of bovine Longissimus dorsi, four Charolaise heifers and four Charolaise bulls muscles were sampled at slaughter and after early and long aging (storage at 2-4 \ub0C for 12 and 26 days respectively). Descriptive sensory evaluation of samples was performed and their tenderness was evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged for 12 and 26 days was analyzed by cartesian and radial 2-D electrophoresis. Student\u2019s t-test and Ranking-PCA analyses were performed to detect proteomic modulation during the different stages of maturation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. Proteomic analysis demonstrated that there were no differences between proteomic patterns of male and females LD muscle. Furthermore, meat maturation caused changes of (in) the abundance of proteins involved in metabolic, structural, and stress related processes. In addition, it must be remarked that the extension of aging beyond 12 days, in the conditions here tested, did not highlight any concrete advantage in terms of sensory quality

2-DE and 2-PE gel electrophoresis.

<p>Representative gel images obtained with 2-DE (panel A) and 2-PE (panel B). Panel C shows the radial map after the Delta2D assisted conversion of coordinates from polar to Cartesian. The lengths of the axes are reported. The highlighted areas of the gels are those reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030911#pone-0030911-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030911#pone-0030911-g003" target="_blank">3</a> as enlarged sections. Sample load: 1.2 mg of total protein in A vs. 120 µg in B.</p