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FONDAZIONE FILARETE PROTEOMIC SERVICES

The proteomic platform consists of: 

Preparation facility lab for the preparation of samples and separation of complex protein mixtures by electrophoretic (1DE and 2DE)

Technological services 

analyses

and chromatographic methodologies. 

Characterization spectrometric,

facility

lab

N-terminal

Technical advise and services for proteomic

for

mass

amino

acid

including

sample

preparation,

proteins separation by electrophoretic and chromatographic methods, mass spectrometry analyses, western blot analyses, N-terminal

sequence and amino acid analysis.

sequence analysis, amino acid analysis and qualitative and quantitative differential analysis

R&D activities

of samples;

The main activities of the platform include: 

Characterization of the proteome of cells and tissues

including

evaluation

of

complex mixtures and characterization of post-

Purification and biochemical characterization of proteins from different sources including

protein

expression, identification of specific proteins in

evaluation of enzymatic activity. 

Renting of equipment for proteomic analyses to customers

translational modifications; 

Analysis of protein-protein interactions and complexes;

Identification of new biomarkers in the clinical and pharmaceutical fields and of antigens for the development of new vaccines by SERPA (serological proteome approach);

Identification of new therapeutic targets for diseases with a high social impact like cancer and neurodegenerative diseases;

of

Viale Ortles 22/4 20139 – Milano (MI) - Italy

Application in nutritional science: screening for

Phone +39 02 566601 Fax +39 02 537250

novel functional bioactives, detection and

Email services@fondazionefilarete.com

Small

molecule

metabolite

profiling

biological samples (metabolomics); 

control

of

food

spoilage

and

allergenic

proteins; 

Development of new protocols for proteomic analysis based on new nanomaterials;

Fondazione Filarete

Quality control of recombinant proteins and synthetic peptides,


1D- and 2Delectrophoresis Technical Specifications: 1)

2)

3)

Ettan™ IPGphor™ 3 IEF System from GE Healthcare Run up to 12 IPG strips (7, 11, 13, 18, or 24 cm) simultaneously PROTEAN II xi | XL Multi-Cells from Biorad Run up to 6 home-made or pre-cast large (up to 18.3 x 19.3 cm) gels Mini-PROTEAN® Tetra Cell from Biorad Run up to 4 home-made or pre-cast mini (7 x 8 cm) gels

Fields of application: 1)

2) 3)

Quantitative studies of gene expression variation and relationships, detection of stages in cellular differentiation and studies of growth cycles, comparative examination of physiological and pathological states for classification and diagnosis of disease, monitoring of drug action and other studies involving global qualitative and quantitative evaluation of protein patterns Determination of MW, subunit composition and polymerization state of a protein Purity evaluation of protein preparations

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

Analytical 2-D gel electrophoresis of surface associated proteins from S. aureus


Western blotting Technical Specifications: 1) 2)

Mini Trans-Blot Cell from Biorad. Transfers two 7.5 x 10 cm gels Trans-Blot Cell from Biorad Transfers two 16 x 20 cm gels

Additional laboratory equipment Proteins blotted on nitrocellulose or polyvinylidendifluoride (PVDF) membranes can be further characterized by immunostaining or N-terminal amino acid sequencing Fields of application 1) 2)

3) 4)

Detection, identification and characterization of proteins following electrophoresis Differential quantitative analysis of expression or post-translational modification levels of proteins from different samples. Detection and characterization of new antigens Probing for biologic activity of proteins, including protein-protein interactions

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it) Evaluation of peripherin nitration in NGF treated PC12 cells


Gel Image Analysis

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

The platform is equipped with a complete set of different gel image acquisition instruments and software allowing tailoring the analysis to specific customers’ needs. All post-electrophoresis staining conditions, including fluorescent dyes, or radioactive-labelled sample, can be detected and quantified over a large dynamic range

Technical Specifications: 1)

Molecular Imager GS-800 Calibrated Densitometer from Biorad Light source Fluorescent: white Wavelength: 400–750 nm Operating modes: Transmissive and reflective Sampling rate: 700 dpi Pixel density: 12-bit Resolution: 36.3 μm

2)

Molecular Imager VersaDoc MP 4000 System from Biorad Excitation wavelengths: Red, green, blue, broadband UV, and white light Emission filters: 530BP, 605BP, 640BP, 695BP Detector: Front-illuminated high-sensitivity CCD with microlens technology Multichannel image collection. Pixel size: 6.8 x 6.8 µm Pixel array size (H x V): 2,184 x 1,472 pixels Pixel data density: 16-bit (0–65,535 levels) Dynamic range: 3.4 orders of magnitude Illumination modes: Trans- and epi-illumination

3)

Molecular Imager ChemiDoc XRS from Biorad Supercooled high-resolution CCD camera with 1.44 megapixel resolution Real-time imaging 16-bit (65,535 gray level) data collection capability Choice of 3 UV transillumination wavelengths

Software available: PD-Quantity One: a package for imaging and analyzing 1-D electrophoretic gels, dot blots, and western blots. Possibility to quantify and analyze a variety of data, including radioactive, chemilumenescent, fluorescent, and color-stained samples acquired from densitometers, storage phosphor imagers, fluorescent imagers, and gel documentation systems. PD-Quest: a package for analysis and databasing of 2D gels. The software offers powerful comparative analysis tools to reveal subtle differences among gels being analyzed (for example, studying the effect of variables such as physiological/pathological conditions, dose-response, and time course).


LC-ESI MASS SPECTROMETRY Technical Specifications: 1)

ThermoFisher LTQ Orbitrap Velos ETD Ion Max™ API source with S-lens ion optics technology Dual-Pressure Linear Ion Trap Orbitrap Mass Analyzer Atmospheric pressure ionization (API) ESI source and μESI source Nanospray source Proxeon Collision cell with axial field gradient Multiple fragmentation techniques: CID, HCD, and ETD Parallel MS and MSn analysis Resolving power of >100,000 Mass accuracy better than 1 ppm

2)

Nano HPLC Ultimate 3000 Flow rate Nano 50-1000 nl/min Flow rate Cap 0.5-10 μl/min Flow rate Micro 10-160 μl/min Pressure range 0.1-50 MPa 6 eluent lines Biocompatible version DGP-3600MB (Titanium) Temperature range 5-85°C 2D HPLC possibility Autosampler WPS-3000

Software available:

Proteome Discoverer:

Sieve:

Bioworks: Prosight:

Promass:

comprehensive proteomics software including several database search engines to complement the breadth of dissociation techniques such as CID, ETD, and HCD for protein identification and PTM characterization, quantization and statistical analysis, automated data processing, for automated, label-free semi-quantitative differential expression analysis of proteins, peptides, and metabolites for protein identification for top down identification and characterization of proteins, including their posttranslational modifications (PTMs) for the analysis of intact proteins and oligonucleotides; optimized for high-throughput applications


Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

MALDI TOF-TOF MASS SPECTROMETRY Technical Specifications: Bruker MALDI TOF-TOF autoflex™ III * MALDI source scoutMTPTM with automatic loading sample Solid-state laser with SmartbeamTM technology, enabling MALDI imaging Linear flight tube (+ and – ions) of 120 cm Reflectron flight tube (+ and – ions) of 215 cm TOF/TOF technology (LIFT™) for fragmentations at high sensibility (LID-LIFT) and high energy (CID) PAN technology for bottom-up and top-down proteomics Patented AnchorChip™ MALDI target technology for fast automation, increased sequence coverage and 10–100 fold sensitivity increase over stainless steel holder Software available: BioTools for protein data analysis FlexImaging for MALDI imaging


Bruker disposable AnchorChip Îą-Tubulin nitrated in rat brain * The MALDI spectrometer is available at CIGA (University of Milano). Researchers of the proteomic platform have free access to this instrument

Fields of application for LC-ESI and MALDI mass spectrometry: General proteomics including: analysis of the complete proteome of cells and tissues, protein identification by fingerprint and MS/MS analysis, molecular weight measurement, evaluation of protein expression, detection and characterization of post-translational modifications, sequence analysis by MS/MS either by PSD, CID, HCD and ETD fragmentation techniques Shotgun LC-MS/MS analysis: direct analysis of complex mixtures to rapidly generate a global profile of the proteic components in the mixture. 2D-LC-MS and MS/MS analysis for the characterization of complex protein samples Label-free quantification of proteins and peptides based on mass spectrometry Clinical Proteomics including qualitative and quantitative differential proteomic analysis of samples, new biomarker discovery and profiling, proteomics of disease and pathogens, evaluation of drug effects on protein patterns SERPA (serological proteome approach) analysis for the identification of antigens and development of new vaccines

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

Network Mapping: identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multi-protein complexes Food analysis by proteomics to authenticate food origin and quality including structural information on unknown ingredients of complex mixtures Polymers analysis: nucleic acids (DNA), peptide nucleic acids (PNA), block copolymers (nanostructures), functionalized polymers, macromolecules Tissue imaging: comprehensive visualization of the spatial distribution of biomarker candidates, drugs and metabolites in a tissue Analysis of low molecular weight compounds. The LC-MS/MS equipment allows to analyze samples previously separated on-line by LC (liquid chromatography). This approach allows the identification and characterization of low molecular weight molecules, including metabolites, drugs and their metabolites, xenobiotics and residues. It is particularly suitable to carry out metabolic profiling to distinguish between normal and alterated states, to study mechanism of drug-induced toxicity and/or efficacy and to discover and identify potential biomarkers.


Automated proteins/peptides sequencer Technical specifications: Applied BioSystems Procise 491 automated protein/peptide sequencer for sequencing intact proteins and peptides based on Edman chemistry, starting from the N-terminal residue. Sample required: at least 40 pmol of protein which can be purified by conventional chromatographic methods or by electrophoresis followed by blotting on commercially available polyvinyliden difluoride (PVDF) membranes. Compatible with most chemicals used in protein science. Number of amino acids identified usually ranging from 10 to 20, depending on the starting material. Longer sequences can be obtained under favourable conditions. Fields of application: Characterization of recombinant proteins: rapid determination of short N-terminal sequence of the intact protein of interest complements and confirms MS-based analyses for quality control of proteins obtained by recombinant-DNA technologies Characterization of N-terminal processing of proteins: removal of Nterminal Met or of longer stretches of amino acid from the Nterminus of proteins are frequently observed both as a result of post-translation modification of proteins from natural sources and during the overexpression and purification of recombinant proteins. This can result in microheterogeneities which must be identified for the complete characterization of the protein of interest. Characterization of proteins from organisms for which no genomic data are available. Edman chemistry always relies on a “de novo� sequencing approach since assignment of the amino acid sequence from experimental results is independent from data-bank searches. Characterization of polypeptide patterns generated by limited proteolysis of the protein of interest to study domain organization, conformational changes and protein-protein interactions. Complementing MS-based determination of molecule weights and N-terminal sequence analysis of fragments separated by SDSPAGE allows a complete description of the proteolytic pattern Local contact: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)


Amino acid analysis Technical specifications: Japan Spectroscopy (Jasco) HPLC equipped with PU980 pumps and 821-FP spectrofluorometer detector. Method: pre-column derivatization of primary amino acids with ortho-phthalaldehyde (OPA) followed by RP-HPLC separation and fluorescence detection Sample amount required: the methods routinely allows quantifying a minimum of 10 pmol of each amino acid, although sensitivity can be lowered but requires careful preliminary handling and preparation of samples by the subject requiring the analysis. Standard error: Âą 5 % Determination of specific non-proteic amino acids (for example for the metabolic profiling of free amino acids in blood) can be performed but may require additional set up of chromatographic conditions for the separation of such amino acids Fields of application: All studies requiring the determination of the exact amino acid content of a sample, such as: -Metabolic profiling of biological fluids such as blood -Amino acid content of food -Exact determination of the protein content of a sample (with a precision unparalleled by any other general method) Local contact: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)


PROTEOMICS