Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

Colomé, Núria; Abián, Joaquín; Aloria, Kerman; Arizmendi, Jesús M.; Barceló-Batllori, Sílvia; Braga-Lagache, Sophie; Burlet‐Schiltz, Odile; Carrascal, Montserrat; Casal, J. Ignacio; Chicano-Gálvez, Eduardo; Chiva, Cristina; Clemente, Luis Felipe; Elortza, Félix; Estanyol, Josep Marı́a; Fernández‐Irigoyen, Joaquín; Fernández-Puente, Patricia; Fidalgo, María José; Froment, Carine; Fuentes, Manuel; Fuentes-Almagro, Carlos; Hainard, Alexandre; Heller, Manfred; Hernández, María Luisa García; Ibarrola, Nieves; Iloro, Ibón; Kieselbach, Thomas; Lario, Antonio; Locard‐Paulet, Marie; Marina-Ramírez, Anabel; Martín, Lucas; Morato-López, Esperanza; Muñoz, Javier; Navajas, Rosana; Odena, María Antonia; Odriozola, Leticia; Oliveira, Eliandre de; Paradela, Alberto; Pasquarello, Carla; Rı́os, Vivian de los; Ruiz-Romero, C.; Sabidó, Eduard; Pino, Manuel M. Sánchez del; Sancho, Jaime; Santamaría, Enrique; Schaeffer-Reiss, Christine; Schneider, Justine; Torre, Carolina De La; Valero, M. Luz; Vilaseca, Marta; Wu, Shuai; Wu, Linfeng; Embún, Pilar Ximénez; Canals, Françesc; Corrales, Fernando J.; ProteoRed-ISCIII,; EuPA,

doi:10.1016/j.jprot.2021.104409

Cited by 3 publications

(2 citation statements)

References 10 publications

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“…In parallel, phosphopeptide enrichment was performed using TiO 2 columns and cleaning of the sample was performed with Oligo R3 reverse-phase before LC-MS analysis. 18 Peptides were solubilized in 2% acetonitrile (ACN) and 0.1% formic acid (FA), and then peptide concentration was determined by Qubit 2.0 Fluorometric Quantitation (Thermo Fisher Scientific). About 1 μg aliquot of each fraction in an injection volume of 5 μL was analyzed by nano LC ESI-MS/MS (Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry) in an Ultimate 3000 nano HPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 240 (Thermo Fisher Scientific).…”

Section: Liquid Chromatography and Mass Spectrometry Analysis (Lc-esi...mentioning

confidence: 99%

Molecular basis of progressive familial intrahepatic cholestasis 3. A proteomics study

Guerrero,

Carmona‐Rodríguez,

Santos

et al. 2024

BioFactors

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Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe rare liver disease that affects between 1/50,000 and 1/100,000 children. In physiological conditions, bile is produced by the liver and stored in the gallbladder, and then it flows to the small intestine to play its role in fat digestion. To prevent tissue damage, bile acids (BAs) are kept in phospholipid micelles. Mutations in phosphatidyl choline transporter ABCB4 (MDR3) lead to intrahepatic accumulation of free BAs that result in liver damage. PFIC3 onset usually occurs at early ages, progresses rapidly, and the prognosis is poor. Currently, besides the palliative use of ursodeoxycholate, the only available treatment for this disease is liver transplantation, which is really challenging for short‐aged patients. To gain insight into the pathogenesis of PFIC3 we have performed an integrated proteomics and phosphoproteomics study in human liver samples to then validate the emerging functional hypotheses in a PFIC3 murine model. We identified 6246 protein groups, 324 proteins among them showing differential expression between control and PFIC3. The phosphoproteomic analysis allowed the identification of 5090 phosphopeptides, from which 215 corresponding to 157 protein groups, were differentially phosphorylated in PFIC3, including MDR3. Regulation of essential cellular processes and structures, such as inflammation, metabolic reprogramming, cytoskeleton and extracellular matrix remodeling, and cell proliferation, were identified as the main drivers of the disease. Our results provide a strong molecular background that significantly contributes to a better understanding of PFIC3 and provides new concepts that might prove useful in the clinical management of patients.

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Section: Liquid Chromatography and Mass Spectrometry Analysis (Lc-esi...mentioning

confidence: 99%

Molecular basis of progressive familial intrahepatic cholestasis 3. A proteomics study

Guerrero,

Carmona‐Rodríguez,

Santos

et al. 2024

BioFactors

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“…Phosphopeptide enrichment was carried out as described previously (58). The TiO2 slurry was prepared by mixing 10 mg Titansphere beads (10 µm, GL Sciences) with 80 µl of 1M glycolic acid in 80% acetonitrile and 1% Trifluoroacetic acid (TFA) for 30 min at 25oC.…”

Section: Methodsmentioning

confidence: 99%

Time-based quantitative proteomic and phosphoproteomic analysis of A549-ACE2 cells during SARS-CoV-2 infection

Milhano dos Santos,

Vindel,

Ciordia

et al. 2024

Preprint

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The outbreak of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2, led to an ongoing pandemic with devastating consequences for the global economy and human health. With the global spread of SARS-CoV-2, multidisciplinary initiatives were launched to explore new diagnostic, therapeutic, and vaccination strategies. From this perspective, proteomics could help to understand the mechanisms associated with SARS-CoV-2 infection and to identify new therapeutic targets for antiviral drug repurposing and/or discovery. A TMT-based quantitative proteomics and phosphoproteomics analysis was performed to study the proteome remodeling of human lung alveolar cells transduced to express human ACE2 (A549-ACE2) after infection with SARS-CoV-2. Targeted PRM analysis was performed to assess the detectability in serum and prognostic value of selected proteins. A total of 6802 proteins and 6428 phospho-sites were identified in A549-ACE2 cells after infection with SARS-CoV-2. Regarding the viral proteome, 8 proteins were differentially expressed after 6 h of infection and reached a steady state after 9 h. In addition, we detected several phosphorylation sites of SARS-CoV-2 proteins, including two novel phosphorylation events at S410 and S416 of the viral nucleoprotein.ImportanceThe differential proteins here identified revealed that A549-ACE2 cells undergo a time-dependent regulation of essential processes, delineating the precise intervention of the cellular machinery by the viral proteins. From this mechanistic background and by applying machine learning modelling, 29 differential proteins were selected and detected in the serum of COVID-19 patients, 14 of which showed promising prognostic capacity. Targeting these proteins and the protein kinases responsible for the reported phosphorylation changes may provide efficient alternative strategies for the clinical management of COVID-19.

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Dynamic Cellular Proteome Remodeling during SARS-CoV-2 Infection. Identification of Plasma Protein Readouts

dos Santos,

Vindel-Alfageme,

Ciordia

et al. 2024

J. Proteome Res.

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Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

Cited by 3 publications

References 10 publications

Molecular basis of progressive familial intrahepatic cholestasis 3. A proteomics study

Molecular basis of progressive familial intrahepatic cholestasis 3. A proteomics study

Time-based quantitative proteomic and phosphoproteomic analysis of A549-ACE2 cells during SARS-CoV-2 infection

Dynamic Cellular Proteome Remodeling during SARS-CoV-2 Infection. Identification of Plasma Protein Readouts

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