Identification of AGR2 partners using mass spectrometry with label-free quantification
| Autoři | |
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| Rok publikování | 2012 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Anterior gradient protein 2 homolog is a member of protein-disulfide isomerase (PDI) family expressed in endoplasmatic reticulum. The suggested function is a co-chaperone aiding the forming of disulfide bonds with role in tumour metastasis. Little knowledge is available about AGR2-interacting or –binding partners in cells. To date, only Mucin 2 was described as the disulfide-linked AGR2 partner and three other proteins reptin, LYPD3 and ?DAG1 were predicted to interact with AGR2 by two-hybrid yeast study. We analysed possible AGR2 partners using cell line with endogenous AGR2 expression (T47D) as well as AGR2-stable transfected cells (H1299). We applied reversible protein cross-linking (DSP agent) on cells in vivo to stabilize protein complexes and precipitated AGR2 with AGR2 specific and un-specific peptides and antibodies followed by Filter Aided Proteome preparation (FASP) and high resolution mass spectrometry analysis of IP eluates (LC-MS/MS on Orbitrap instrument). Label-Free Quantification (LFQ) of proteins identified in Swissprot/Uniprot database was performed in MaxQuant data analysis software. Applying described method with elimination of un-specifically bound proteins we obtained AGR2 partners in AGR2-specific IP eluates. PDI members A3 and A6 were repeatedly identified in both cell lines, while AGR3 protein was found only in T47D cells based on different expression in cell lines, quantitatively the proteins are significantly presented in higher levels in cross-linked samples in comparison to un-cross-linked control. |
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