Molecularly Imprinted Polymers for characterization of rare phosphorylation events

Ignacio Arribas Díez, Anil Incel, Chau Huynh

Research output: Contribution to conference without publisher/journalPosterResearch

Abstract

Protein phosphorylation and cell signaling networks have been studied thoroughly by large-scale phosphoproteomics technologies, including affinity enrichment methods and LC-MS/MS. A vast majority of studies reported phosphorylation of Ser, Thr and Tyr residues in proteins based on immobilized metal affinity chromatography, i.e. IMAC and TiO2 (pS, pT, pY) and antibody-based enrichment (pY). Recently, we reported the application of molecularly imprinted polymers (MIPs) for pY-peptide enrichment (Bllaci et al., 2017). Here, we report initial results obtained by using a novel monolithic MIP capillary column directed towards phosphohistidine (pHis) residues and a second MIP for targeting specific pY motifs. Phosphohistidine residues are labile at acidic conditions necessitating special precautions during sample preparation and LC-MS analysis. We used chemical phosphorylation using potassium phosphoramidate (Wei et al. 1991) to generate myoglobin pHis-peptides, which were used for testing the crushed monolith and column MIP by LC-MS/MS. Peptides from ZAP70 kinase were spiked on a 12 proteins digest for characterizing the pY-motif specific MIP. This method was performed on batch mode with different spiking levels to address the sensitivity of the MIP by LC-MS/MS analysis of the elution fractions. We will present this new modification-specific proteomics strategy and preliminary results obtained by using MIPs in combination with LC-MS/MS for biomolecule characterization.
Original languageEnglish
Publication date2019
Publication statusPublished - 2019
EventProteomics in Cell Biology and Disease Mechanisms: EMBL–Wellcome Genome Campus Conference - EMBL Advanced Training Centre, Heidelberg, Germany
Duration: 7. Mar 20199. Mar 2019
https://www.embl.de/training/events/2019/PRO19-02/programme/index.html

Conference

ConferenceProteomics in Cell Biology and Disease Mechanisms
LocationEMBL Advanced Training Centre
Country/TerritoryGermany
CityHeidelberg
Period07/03/201909/03/2019
Internet address

Keywords

  • MIPs
  • Phosphorylation
  • Mass spectrometry
  • Proteomics

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