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en:aree:proteomica:ms:start [2018/08/10 15:19] Gianluca Frustagli English translation. |
en:aree:proteomica:ms:start [2023/06/22 12:43] (current) Gianluca Frustagli Update of text regarding new instruments. |
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| - | {{:aree:proteomica:ms:lab.jpeg?400 |}} The unit is equipped with an **LTQ XL Ion Trap** mass spectrometer from ThermoFisher with an additional ETD (Electron Transfer Dissociation) fragmentation(([[http://www.pnas.org/cgi/content/full/101/26/9528|Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry]])). | + | {{:aree:proteomica:ms:lab.jpeg?400 |}} Two **Tribrid Orbitrap Fusion** high resolution mass spectrometers are present in the MS Unit. The Tribrid architecture combines three mass analyzers: a quadrupole, an ion trap and an Orbitrap. Multiple fragmentation techniques are adopted: CID (Collision Induced Dissociation), HCD (Higher-energy Collisional Dissociation) and ETD (Electron transfer dissociation)(([[http://www.pnas.org/cgi/content/full/101/26/9528|Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry]])). |
| - | This instrument, in-line with a nano-capillary chromatograph, allows the separation of peptide mixtures with different complexity degree and the subsequent identification of the constituent proteins through dedicated software (LC-MS / MS analysis). This instrumentation is of great support in the biomedical research projects that require the identification and/or characterization of proteins and their level of expression. The samples to be analyzed can be multiple: synthetic proteins, semi-purified proteins, immuno-precipitation, sub-proteomes, total proteomes. Generally, the LC-MS / MS analysis is preceded by electrophoresis (PAGE) for a qualitative and quantitative estimate of the sample followed by protein in-gel digestion. | + | A FAIMS interface //(field asymmetric-waveform ion-mobility spectrometry)// is also available. The spectrometers, on line with nano-capillary chromatographs, allow the separation of more or less complex peptide mixtures and the subsequent identification of the constituent proteins, through dedicated software. This instrumentation is of great support in those biomedical research projects in which one is interested in the identification and/or characterization of proteins and their level of expression. The samples to analyze can be many: synthetic proteins, semi-purified proteins, immuno-precipitations, sub-proteomes, total proteomes. Generally, the LC-MS/MS analysis is preceded by protein mixture in solution digestion(([[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1322.html|Universal sample preparation method for proteome analysis]])) or protein separation by electrophoresis (PAGE) followed by in-gel digestion, in order to carry out a qualitative and quantitative estimation of the sample. |
| - | In case of very poor samples or for methodological needs, the digestion can be carried out in solution and followed by off-line chromatography before the LC-MS /MS analysis(([[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1322.html|Universal sample preparation method for proteome analysis]])). In comparative studies, relative quantitative determination is possible through isotopic labeling techniques(([[https://www.ncbi.nlm.nih.gov/pubmed/17902639|iTRAQ Reagent-Based Quantitative Proteomic Analysis on a Linear Ion Trap Mass Spectrometer]]))(([[http://www.neurology.org/content/79/10/1012.abstract|Increased levels of acute-phase inflammatory proteins in plasma of patients with sporadic CJD]])) or label-free(([[https://www.ncbi.nlm.nih.gov/pubmed/23794183|Critical assessment of proteome-wide label-free absolute abundance estimation strategies]])). The unit relies particularly on extensive experience in the following areas: | + | In comparative studies it is possible to determine the relative quantity using isotope labeling(([[https://www.ncbi.nlm.nih.gov/pubmed/17902639|iTRAQ Reagent-Based Quantitative Proteomic Analysis on a Linear Ion Trap Mass Spectrometer]]))(([[http://www.neurology.org/content/79/10/1012.abstract|Increased levels of acute-phase inflammatory proteins in plasma of patients with sporadic CJD]])) or label-free(([[https://www.ncbi.nlm.nih.gov/pubmed/23794183|Critical assessment of proteome-wide label-free absolute abundance estimation strategies]])) techniques. In particular, the unit draws on extensive experience in the following areas: |
| - | * preparation and characterization, also by means of quantitative comparative analysis, of sub-proteomes extracted from cultured cells, fluids and other biological tissues; | + | * preparation and characterization, also through quantitative comparative analysis, of sub-proteomes extracted from cultured cells, fluids and other biological tissues; |
| - | * analysis of membrane microdomains (rafts) and extra-cellular vesicles (exosomes and microparticles); | + | * analysis of membrane microdomains (rafts) and extracellular vesicles (exosomes and microparticles); |
| - | * protein characterization: protein sequence analysis by combining different enzymes, post-translational modifications, N-termini and neo-N-termini determination by chemical derivatization, analysis of protein adducts with small molecules and/or drugs; | + | * characterization of proteins: analysis of the protein sequence by combining different enzymes, study of post-translational modifications, determination of the N-terminus and neo-N-termini by chemical derivatization, formation of adducts between proteins and small molecules and/or drugs; |
| * interactomics by analysis of immunoprecipitates; | * interactomics by analysis of immunoprecipitates; | ||
| * quantitative analysis of specific peptides in mixture. | * quantitative analysis of specific peptides in mixture. | ||
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| Several bioinformatic analysis approaches are available for the analysis of results. | Several bioinformatic analysis approaches are available for the analysis of results. | ||
| - | <wrap em>Service requests to this unit must be sent by filling out the '[[richiesta|Service Request]]' online form. You will be contacted for a preliminary meeting.</wrap> | + | \\ |
| + | |||
| + | === To ask for the service: === | ||
| + | |||
| + | - preliminary contact the [[chi_siamo|MS unit staff]]; | ||
| + | - fill out the online form '**[[richiesta|Service Request]]**' briefly illustrating the scientific problem, type and number of samples etc.; | ||
| + | - plan a meeting with the unit staff to discuss methods of analysis, costs, timing etc. | ||
| <wrap em>We recommend that you follow the advices in the '[[linee_guida|Guidelines]]' section during sample preparation.</wrap> | <wrap em>We recommend that you follow the advices in the '[[linee_guida|Guidelines]]' section during sample preparation.</wrap> | ||
| - | > Contact: [[marta.ponzi@iss.it|Marta Ponzi]] | + | \\ |
| - | > ☎ **+39 06 4990 3134** | + | |
| + | > Contact: [[serena.camerini@iss.it|Serena Camerini]] | ||
| + | > ☎ **+39 06 4990 3164** | ||
| + | > Fax **+39 06 4990 2040** | ||
| > Where we are: building **30**, floor **A**, room **11** | > Where we are: building **30**, floor **A**, room **11** | ||
| - | > FIXME **To be revised.**\\ //(remove this paragraph finished)// | + | /* > FIXME **To be revised.**\\ //(remove this paragraph finished)// */ |
