Core facilities
PROMEFA
Mission
The Proteomics and Metabolomics Facility (ProMeFa) provides different analytical approaches aimed at structurally characterizing small molecules, peptides and proteins. The Facility is equipped with state-of-the-art mass spectrometers and has access to the nuclear magnetic resonance (NMR) instrument within the Consorzio 7C (Ronzoni Istitute for Chemical and Biochemical Research, Milan). Mass spectrometry and NMR are crucial techniques, which give a major contribution to the description and understanding of biological processes at the molecular level.
Services
The goal of the Facility is to assist scientists in their research on a fee-for-service basis, covering all the operating costs, including consumables, time-machine and technical support.
PROMIFA services can be divided in:
- Biochemical Services
- Mass Spectrometry Services
- Proteomics Services
- Metabolomics Services
All the services comprise appropriate scientific consultancy, interpretation of the results, a report on the analytical data signed by the facility manager.
Guidelines and detailed protocols for sample preparation are available here
Request form for internal and external users are available here:
Access info
Due to the complexity and cost of some of the equipment, only the staff of the facility has direct access to the machines. Interested people can be trained upon request. Samples are accepted every day.
ProMeFa is located in Dibit 2, San Gabriele 2, C1, 4th floor (mass spectrometry services) and at Dibit 2, Basilica A3, 3rd floor (NMR and protein biochemistry services).
First time users and anyone wanting to submit large numbers of samples at once (more than 15) are urged to contact the Facility prior to sample submission. The sample(s) has to be shipped together with the filled request form/order. PROMIFA strongly suggest contacting the staff of the Facility before filling in any request form, in order to define exactly the activity to be carried out on the samples.
Here you can find some frequently asked questions: FAQs.
Mass Spectrometry analysis
- Protein characterization:
- sample purification (zip tip or similar) and intact protein/peptide MW determination by nanospray off line MS and MS/MS.
- Protein identification: PMF and sequencing
- PTM-characterization: validation by Western Blotting and 2DE +/- removal of the PTM (glycosylation, phosphorylation);
- phosphorylation characterization by TiO2 or IMAC enrichment and mass spectrometry;
- identification of a spot or band by in gel digestion and LC-MS/MS.
- Quantitative protein profiling by mass spectrometry and statistical analysis. Differential protein display in two or more diverse conditions using:
- iTRAQ or TMT labelling of peptides,
- SILAC methodology,
- Label-free approach.
- Biomarker profiling in biological samples by 2DE, image analysis and statistical evaluation. Differential protein display in two or more diverse conditions in plasma, serum, urine, tissue.
- Metabolomics/Lipidomics by mass spectrometry and statistical analysis. Untargeted workflow for differential small molecules/lipids display in: plasma, serum, urine, tissues.
- Quantitative Metabolomics/Lipidomics by mass spectrometry and statistical analysis. Targeted workflow for specific small molecules/lipids display in plasma, serum, urine, tissues.
Nuclear magnetic resonance (NMR) analysis
- NMR spectra acquisition (1D 1H spectra for profiling, untargeted and targeted analysis; 2D spectra to assist metabolites identification from tissue/cells for endometabolomic analysis, biofluids, extracellular medium
- Statistical analysis: Multi- and univariate statistical analysis (minimum 3 samples for group, 2 groups) and pathway analysis for untargeted approach
- Targeted analysis: Profiling and semi quantification using Chenomx 7.7 and univariate statistical analysis (minimum 3 samples for group, 2 groups)
- Stable Isotope Resolved Metabolomics (SIRM) for metabolic flux analysis
- Unknown metabolites identification
Protein biochemistry analysis (available either as service or for a supervised use only for internal laboratories)
- Protein purification by fast protein liquid chromatography (FPLC): size exclusion chromatography; ion exchange chromatography; hydrophobic interaction chromatography; affinity chromatography.
- Protein detection by capillary Western blot: protein immune-detection and semi-quantitative evaluation of protein expression in biological samples.
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PROTEIN ANALYSIS BY MASS SPECTROMETRY |
INTERNAL/EXTERNAL |
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Sample purification (zip tip or similar) and intact protein/peptide MW determination by nanospray off line MS |
75 / 82 € |
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Sample purification (zip tip or similar) and intact protein/peptide MW determination by nanospray off line MS/MS |
100/110 € |
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Protein identification: PMF and sequencing |
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PTM-characterization: validation by Western Blotting and 2DE +/- removal of the PTM (glycosylation, phosphorylation) |
500/600 € |
|
Phosphorylation characterization by TiO2 or IMAC enrichment and mass spectrometry |
300/350 € |
|
Identification of a spot or band by in gel digestion and LC-MS/MS |
200/240 € (the price goes down with the number of bands/spots) |
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Biomarker profiling in biological samples by 2DE, image analysis and statistical evaluation |
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Differential protein display in control vs. patient biological samples: plasma, serum, urine, tissue. Six samples: control vs. treated/patient in triplicates |
1500/1800 € (800/960 € for each additional condition) |
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Quantitative protein profiling by mass spectrometry and statistical analysis |
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iTRAQ or TMT labelling of peptides (run in technical duplicate) |
2500/3000 € |
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SILAC methodology (run in technical duplicate) |
2500/3000 € |
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Label-free approach (run in technical triplicate) (iTRAQ or TMT-reagents or labelled amino acids are in charge to the customers) |
800/960 € |
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Metabolomics/Lipidomics by mass spectrometry and statistical analysis |
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Untargeted workflow for differential small molecules/lipids display in: plasma, serum, urine, tissues (C18, HILIC, C8, positive and negative polarity). |
Prices are tailored to each project |
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Quantitative Metabolomics/Lipidomics by mass spectrometry and statistical analysis |
|
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Targeted workflow for specific small molecules/lipids displays in: plasma, serum, urine, tissues |
Prices are tailored to each project |
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Access to MS |
15/25 € per hour |
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Data analysis |
60/80 € per hour |
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METABOLOMICS ANALYSIS BY NMR |
INTERNAL/EXTERNAL |
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NMR experiment 1D 1H or 2D NMR experiments (tissue/cells) |
37/45 € per sample |
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NMR experiment 1D 1H or 2D NMR experiments (biofluids) |
30/38 € per sample |
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Multivariate statistical analysis |
60/80 € per hour |
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Targeted analysis and semi quantification using Chenomx 7.7 |
30/50 € per sample |
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Data analysis (pathways analysis) |
60/80 € per hour |
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Unknown metabolites identification |
Price is tailored after discussion |
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Stable Isotope Resolved Metabolomics (SIRM) (fluxomics) |
Price is tailored after discussion |
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PROTEIN BIOCHEMISTRY ANALYSIS |
INTERNAL/EXTERNAL |
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Protein Purification by FPLC |
Price is tailored after discussion |
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Protein detection by capillary Western blot |
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Protein immune-detection – 1 cartridge 13 samples |
150/200 € |
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Protein immune-detection – 1 cartridge 13 samples |
200/250 € |
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Semi-quantitative evaluation of protein expression – 1 cartridge 13 samples |
250/320 € |
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Semi-quantitative evaluation of protein expression – 1 cartridge 25 samples |
320/400 € |
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Data analysis with Compass® software |
60/80 € per hour |
ACTIVITIES |
PROCESSING TIME |
| Protein characterization | |
| Sample purification (zip tip or similar) and intact protein MW determination by MALDI or nanospray off line MS or MS/MS | 3 days |
| Protein identification: PMF and sequencing | |
| PTM-characterization: validation by Western Blotting and 2DE +/- removal of the PTM (glycosylation, phosphorylation) | 2 weeks |
| Phosphorylation characterization by TiO2 or IMAC enrichment and mass spectrometry | 2 weeks |
|
Identification of a spot or band by in gel digestion and LC-MS/MS |
1 week |
Processing timing depends on the number of samples for:
- Biomarker profiling in biological samples by 2DE, image analysis and statistical evaluation. Differential protein display in two or more diverse conditions in: plasma; serum; urine; tissues.
- Quantitative protein profiling by mass spectrometry and statistical analysis. Differential protein display in two or more diverse conditions using: iTRAQ or TMT labelling of peptide; SILAC methodology;Label-fre approach.
- Metabolomics/Lipidomics by mass spectrometry and statistical analysis. Untargeted workflow for differential small molecules/lipids display in: plasma; serum; urine; tissues.
- Quantitative Metabolomics/Lipidomics by mass spectrometry and statistical analysis. Targeted workflow for specific small molecules/lipids display in: plasma; serum; urine; tissues.
NMR metabolomics analysis
ACTIVITIES |
PROCESSING TIME |
| Untargeted analysis (samples preparation, experiments acquisition and multivariate analysis) | 2-3 weeks |
| Targeted analysis (sample preparation, experiments acquisition, identification of metabolites by chenomx and 2D NMR experiments, statistical analysis) | 2-4 weeks |
Protein biochemistry analysis
ACTIVITIES |
PROCESSING TIME |
| Protein Purification by FPLC | 1 weeks |
| Protein detection by capillary Western blot |
|
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3 days |
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3 days |
|
1 week |
Output of the results will be sent in an Excel or Word format, along with information on: mass spectrometer settings, protein database version, search parameters, experimental conditions for 2DE, constraints for image analysis and statistical evaluation.In case of maintenance or out of order instrumentation as well as when multiple samples are submitted, the processing time can be longer.
When the Facility will send the results of the analysis (by fax or e-mail) then the Grant Office will give the invoice to the user for the payment.
Barbariga M, Vallone F, Mosca E, Bignami F, Magagnotti C, Fonteyne P, Chiappori F, Milanesi L, Rama P, Andolfo A, Ferrari G. The role of extracellular matrix in mouse and human corneal neovascularization.Sci Rep. 2019 Oct 3;9(1):14272. doi: 10.1038/s41598-019-50718-8.
Lenti E, Bianchessi S, Proulx ST, Palano MT, Genovese L, Raccosta L, Spinelli A, Drago D, Andolfo A, Alfano M, Petrova TV, Mukenge S, Russo V, Brendolan A. Therapeutic Regeneration of Lymphatic and Immune Cell Functions upon Lympho-organoid Transplantation. Stem Cell Reports. 2019 Jun 11;12(6):1260-1268. doi: 10.1016/j.stemcr.2019.04.021. Epub 2019 May 30.
Alfano M, Pederzoli F, Locatelli I, Ippolito S, Longhi E, Zerbi P, Ferrari M, Brendolan A, Montorsi F, Drago D, Andolfo A, Nebuloni M, Salonia A. Impaired testicular signaling of vitamin A and vitamin K contributes to the aberrant composition of the extracellular matrix in idiopathic germ cell aplasia. Fertil Steril. 2019 Apr;111(4):687-698. doi: 10.1016/j.fertnstert.2018.12.002.
Magagnotti C, Zerbini G, Fermo I, Carletti RM, Bonfanti R, Vallone F, Andolfo A. Identification of nephropathy predictors in urine from children with a recent diagnosis of type 1 diabetes. J Proteomics. 2019 Feb 20;193:205-216. doi: 10.1016/j.jprot.2018.10.010. Epub 2018 Oct 24.
Ferrari A, Longo R, Fiorino E, Silva R, Mitro N, Cermenati G, Gilardi F, Desvergne B, Andolfo A, Magagnotti C, Caruso D, Fabiani E, Hiebert SW, Crestani M. HDAC3 is a molecular brake of the metabolic switch supporting white adipose tissue browning. Nat Commun. 2017 Jul 21;8(1):93. doi: 10.1038/s41467-017-00182-7.
Drago D, Basso V, Gaude E, Volpe G, Peruzzotti-Jametti L, Bachi A, Musco G, Andolfo A, Frezza C, Mondino A, Pluchino S. Metabolic determinants of the immune modulatory function of neural stem cells. J Neuroinflammation. 2016 Sep 2;13(1):232. doi: 10.1186/s12974-016-0667-7.
Nebuloni M, Albarello L, Andolfo A, Magagnotti C, Genovese L, Locatelli I, Tonon G, Longhi E, Zerbi P, Allevi R, Podestà A, Puricelli L, Milani P, Soldarini A, Salonia A, Alfano M. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix. Sci Rep. 2016 Mar 4;6:22522. doi: 10.1038/srep22522.
Brunati M, Perucca S, Han L, Cattaneo A, Consolato F, Andolfo A, Schaeffer C, Olinger E, Peng J, Santambrogio S, Perrier R, Li S, Bokhove M, Bachi A, Hummler E, Devuyst O, Wu Q, Jovine L, Rampoldi L. The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin. Elife. 2015 Dec 17;4:e08887. doi: 10.7554/eLife.08887.
Barbariga M, Curnis F, Andolfo A, Zanardi A, Lazzaro M, Conti A, Magnani G, Volontè MA, Ferrari L, Comi G, Corti A, Alessio M. Ceruloplasmin functional changes in Parkinson's disease-cerebrospinal fluid Mol Neurodegener. 2015 Nov 4;10:59. doi: 10.1186/s13024-015-0055-2.
Drusian L, Nigro EA, Mannella V, Pagliarini R, Pema M, Costa ASH, Benigni F, Larcher A, Chiaravalli M, Gaude E, Montorsi F, Capitanio U, Musco G, Frezza C, Boletta A. mTORC1 Upregulation Leads to Accumulation of the Oncometabolite Fumarate in a Mouse Model of Renal Cell Carcinoma. Cell Rep. 2018 Jul 31;24(5):1093-1104.e6. doi: 10.1016/j.celrep.2018.06.106.
Chiaravalli M, Rowe I, Mannella V, Quilici G, Canu T, Bianchi V, Gurgone A, Antunes S, D'Adamo P, Esposito A, Musco G, Boletta A. 2-Deoxy-d-Glucose Ameliorates PKD Progression. J Am Soc Nephrol. 2016 Jul;27(7):1958-69. doi: 10.1681/ASN.2015030231. Epub 2015 Nov 3.
Rowe I, Chiaravalli M, Mannella V, Ulisse V, Quilici G, Pema M, Song XW, Xu H, Mari S, Qian F, Pei Y, Musco G, Boletta A. Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med. 2013 Apr;19(4):488-93. doi: 10.1038/nm.3092. Epub 2013 Mar 24.
