Identification of AGR2 partners using mass spectrometry with
label-free quantification

a 2012

Identification of AGR2 partners using mass spectrometry with label-free quantification

BOUCHALOVÁ, Pavla, Pavel BOUCHAL, Alexander SCHERL, David POTĚŠIL, Lenka HERNYCHOVÁ et. al.

Basic information

Original name

Identification of AGR2 partners using mass spectrometry with label-free quantification

Authors

BOUCHALOVÁ, Pavla, Pavel BOUCHAL, Alexander SCHERL, David POTĚŠIL, Lenka HERNYCHOVÁ, Bořivoj VOJTĚŠEK and Roman HRSTKA

Edition

3rd RECAMO joint meeting: Role of p53, MDM2, AGR2/3 and ubiquitin/chaperone systems in tumour biology, 2012

Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10600 1.6 Biological sciences

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

Organization unit

Faculty of Science

Změněno: 16/8/2016 15:17, doc. Mgr. Pavel Bouchal, Ph.D.

Abstract

V originále

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.

Links

GAP304/10/0868, research and development project

Name: Studium molekulárních mechanismů časného metastazování do lymfatických uzlin u karcinomu prsu nízkého stupně malignity pomocí proteomických technik

Investor: Czech Science Foundation

Citovat

BOUCHALOVÁ, Pavla, Pavel BOUCHAL, Alexander SCHERL, David POTĚŠIL, Lenka HERNYCHOVÁ, Bořivoj VOJTĚŠEK and Roman HRSTKA. Identification of AGR2 partners using mass spectrometry with label-free quantification. In 3rd RECAMO joint meeting: Role of p53, MDM2, AGR2/3 and ubiquitin/chaperone systems in tumour biology. 2012.

@proceedings{1351749,
   author = {Bouchalová, Pavla and Bouchal, Pavel and Scherl, Alexander and Potěšil, David and Hernychová, Lenka and Vojtěšek, Bořivoj and Hrstka, Roman},
   booktitle = {3rd RECAMO joint meeting: Role of p53, MDM2, AGR2/3 and ubiquitin/chaperone systems in tumour biology},
   language = {eng},
   title = {Identification of AGR2 partners using mass spectrometry with label-free quantification},
   year = {2012}
}

TY  - CONF
ID  - 1351749
AU  - Bouchalová, Pavla - Bouchal, Pavel - Scherl, Alexander - Potěšil, David - Hernychová, Lenka - Vojtěšek, Bořivoj - Hrstka, Roman
PY  - 2012
TI  - Identification of AGR2 partners using mass spectrometry with label-free quantification
N2  - 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.
ER  -

BOUCHALOVÁ, Pavla, Pavel BOUCHAL, Alexander SCHERL, David POTĚŠIL, Lenka HERNYCHOVÁ, Bořivoj VOJTĚŠEK and Roman HRSTKA. Identification of AGR2 partners using mass spectrometry with label-free quantification. In \textit{3rd RECAMO joint meeting: Role of p53, MDM2, AGR2/3 and ubiquitin/chaperone systems in tumour biology}. 2012.

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