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. 

Proteomics: 

• Identification of a spot or a band by in gel digestion and LC-MS/MS  

• PTM-characterization in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces: 

• glycosylation by enzymatic digestion followed by LC-MS/MS  

• acetylation by PTMScan enrichment (Cell Signaling Technologies) followed by LC-MS/MS 

• phosphorylation (Tyr/Thr/Ser)  by TiO2 or IMAC enrichment followed by LC-MS/MS 

• Quantitative protein profiling by mass spectrometry and statistical analysis. Differential protein display in two or more diverse conditions in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces, using: 

• iTRAQ or TMT labelling of peptides 

• SILAC methodology 

• Label-free approach 

Metabolomics/Lipidomics:  

• Untargeted workflow for differential small molecules/lipids display in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces. 

• Targeted workflow for specific small molecules/lipids display in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces. 

• Tracing experiments in cell culture using stable isotope labelled (13C/15N) small molecules (glucose, glutamine, arginine)  

 

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  8.4 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 detection by capillary Western blot: protein immune-detection and semi-quantitative evaluation of protein expression in biological samples. 

ANALYSIS BY MASS SPECTROMETRY	INTERNAL/EXTERNAL
Protein Characterization
Sample purification (zip tip or similar) and intact protein/peptide MW determination by nanospray off line MS	75 / 82 €
Sample purification (zip tip or similar) and intact protein/peptide MW determination by nanospray off line MS/MS	100/110 €
Proteomics
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)
PTM-characterization in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces: glycosylation by enzymatic digestion followed by LC-MS/MS* acetylation by PTMScan enrichment (Cell Signaling Technologies) followed by LC-MS/MS* phosphorylation (Tyr/Thr/Ser) by TiO2 or IMAC enrichment followed by LC-MS/MS*	300/350 €
Prices are tailored to each project; *300/350 € per single protein
Quantitative protein profiling by mass spectrometry and statistical analysis. Differential protein display in two or more diverse conditions in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces, using:
iTRAQ* or TMT* labelling of peptides (run in technical duplicate)	2500/3000 €
SILAC methodology* (run in technical duplicate)	2500/3000 €
Label-free approach (run in technical triplicate)	800/960 €
*iTRAQ or TMT-reagents or labelled amino acids are in charge to the customers
Metabolomics/Lipidomics
Untargeted workflow for differential small molecules/lipids display in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces Targeted workflow for specific small molecules/lipids display in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces. Tracing experiments in cell culture stable isotope labelled (13C/15N) small molecules (glucose, glutamine, arginine) Prices are tailored to each project
Data analysis	60/80 € per hour

 

METABOLOMICS ANALYSIS BY NMR	INTERNAL/EXTERNAL
NMR experiment 1D 1H or 2D NMR experiments (tissue/cells)	37/45 € per sample
NMR experiment 1D 1H or 2D NMR experiments (biofluids)	30/38 € per sample
Multivariate statistical analysis	60/80 € per hour
Targeted analysis and semi quantification using Chenomx 8.4	30/50 € per sample
Data analysis (pathways analysis)	60/80 € per hour
Unknown metabolites identification	Price is tailored after discussion
Stable Isotope Resolved Metabolomics (SIRM) (fluxomics)	Price is tailored after discussion

 

PROTEIN BIOCHEMISTRY ANALYSIS
Protein detection by capillary Western blot (For the fees, please contact Dr. Mannella: mannella.valeria@hsr.it)
Protein immune-detection – 1 cartridge 13 samples
Protein immune-detection – 1 cartridge 13 samples
Semi-quantitative evaluation of protein expression – 1 cartridge 13 samples
Semi-quantitative evaluation of protein expression – 1 cartridge 25 samples
Data analysis with Compass® software

 

MASS SPECTROMETRY ANALYSIS 

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
Proteomics:
Identification of a spot or a band by in gel digestion and LC-MS/MS	1 week per single protein

PROCESSING TIMING DEPENDS ON THE NUMBER OF SAMPLES FOR:

           Proteomics: 

• PTM-characterization in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces: 

  • glycosylation by enzymatic digestion followed by LC-MS/MS  

  • acetylation by PTMScan enrichment (Cell Signaling Technologies) followed by LC-MS/MS phosphorylation (Tyr/Thr/Ser)  by TiO2 or IMAC enrichment followed by LC-MS/MS  

• Quantitative protein profiling by mass spectrometry and statistical analysis. Differential protein display in two or more diverse conditions in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces, using: 

  • iTRAQ or TMT labelling of peptides 

  • SILAC methodology 

  • Label-free approach 

Metabolomics/Lipidomics:

Untargeted workflow for differential small molecules/lipids display in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces.  Targeted workflow for specific small molecules/lipids display in several biological fluids (plasma, serum, urine, CSF), cells and conditioned media, tissues, feces.  Tracing experiments in cell culture using stable isotope labelled (13C/15N) small molecules (glucose, glutamine, arginine)

 

NMR METABOLOMICS ANALYSIS

ACTIVITIES	PROCESSING TIME
Untargeted analysis (samples preparation, experiments acquisition and multivariate analysis)	4-6 weeks
Targeted analysis (sample preparation, experiments acquisition, identification of metabolites by chenomx and 2D NMR experiments, statistical analysis)	4-6 weeks

Processing time depends on the number of samples and type of analysis.

 

PROTEIN BIOCHEMISTRY ANALYSIS

ACTIVITIES	PROCESSING TIME
Protein detection by capillary Western blot
Protein immune-detection	3 days
Semi-quantitative evaluation of protein expression	3 days
Data analysis with Compass® software	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.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.

Anderluzzi G, Ghitti M, Gasparri AM, Taiè G, Sacchi A, Gori A, Andolfo A, Pozzi F, Musco G, Curnis F, Corti A. A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins. Cell Mol Life Sci. 2024 Jan 30;81(1):68. doi: 10.1007/s00018-023-05102-1. 

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Lazzarato L, Bianchi L, Andolfo A, Granata A, Lombardi M, Sinelli M, Rolando B, Carini M, Corsini A, Fruttero R, Arnaboldi L. Proteomics Studies Suggest That Nitric Oxide Donor Furoxans Inhibit In Vitro Vascular Smooth Muscle Cell Proliferation by Nitric Oxide-Independent Mechanisms. Molecules. 2023 Jul 28;28(15):5724. doi: 0.3390/molecules28155724. 

Mocciaro E, Giambruno R, Micheloni S, Cernilogar FM, Andolfo A, Consonni C, Pannese M, Ferri G, Runfola V, Schotta G, Gabellini D. WDR5 is required for DUX4 expression and its pathological effects in FSHD muscular dystrophy. Nucleic Acids Res. 2023 Jun 9;51(10):5144-5161. doi: 10.1093/nar/gkad230. 

Panebianco C, Pisati F, Villani A, Andolfo A, Ulaszewska M, Bellini E, Ferro C, Lombardi R, Orsenigo F, Latiano TP, Belmonte B, Tripodo C, Perri F, Pazienza V. Counteracting gemcitabine+nab-paclitaxel induced dysbiosis in KRAS wild type and KRASG12D mutated pancreatic cancer in vivo model. Cell Death Discov. 2023 Apr 5;9(1):116. doi: 10.1038/s41420-023-01397-y. 

Lo Conte M, Antonini Cencicchio M, Ulaszewska M, Nobili A, Cosorich I, Ferrarese R, Massimino L, Andolfo A, Ungaro F, Mancini N, Falcone M. A diet enriched in omega-3 PUFA and inulin prevents type 1 diabetes by restoring gut barrier integrity and immune homeostasis in NOD mice. Front Immunol. 2023 Jan 13;13:1089987. doi: 10.3389/fimmu.2022.1089987.  

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Pieri V, Gallotti AL, Drago D, Cominelli M, Pagano I, Conti V, Valtorta S, Coliva A, Lago S, Michelatti D, Massimino L, Ungaro F, Perani L, Spinelli A, Castellano A, Falini A, Zippo A, Poliani PL, Moresco RM, Andolfo A, Galli R. Aberrant L-Fucose Accumulation and Increased Core Fucosylation Are Metabolic Liabilities in Mesenchymal Glioblastoma. Cancer Res. 2023 Jan 18;83(2):195-218. doi: 10.1158/0008-5472.CAN-22-0677. 

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Panebianco C, Villani A, Pisati F, Orsenigo F, Ulaszewska M, Latiano TP, Potenza A, Andolfo A, Terracciano F, Tripodo C, Perri F, Pazienza V. Butyrate, A postbiotic of intestinal bacteria, affects pancreatic cancer and gemcitabine response in in vitro and in vivo models. Biomed Pharmacother. 2022 Jul;151:113163. doi: 10.1016/j.biopha.2022.113163.   

Panebianco C, Pisati F, Ulaszewska M, Andolfo A, Villani A, Federici F, Laura M, Rizzi E, Potenza A, Latiano TP, Perri F, Tripodo C, Pazienza V. Tuning gut microbiota through a probiotic blend in gemcitabine-treated pancreatic cancer xenografted mice. Clin Transl Med. 2021 Nov;11(11):e580. doi: 10.1002/ctm2.580. 

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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.