Projects per year
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.
|Publication status||Published - 2019|
|Event||Proteomics in Cell Biology and Disease Mechanisms: EMBL–Wellcome Genome Campus Conference - EMBL Advanced Training Centre, Heidelberg, Germany|
Duration: 7. Mar 2019 → 9. Mar 2019
|Conference||Proteomics in Cell Biology and Disease Mechanisms|
|Location||EMBL Advanced Training Centre|
|Period||07/03/2019 → 09/03/2019|
- Mass spectrometry
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- 1 Finished
01/11/2016 → 31/10/2020