Chapter 2
Identification of PDZ Interactions by Affinity Purification
and Mass Spectrometry Analysis
Avais M. Daulat, Ste ´ phane Audebert, Mo ˆ nica Wagner, Luc Camoin,
and Jean-Paul Borg
Abstract
Identification of protein networks becomes indispensable for determining the function of a given protein of
interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This
motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and
maintenance of cell architecture. In the present chapter, we present two complementary approaches
(immunopurification and peptide-based purification procedures) followed by mass spectrometry analysis
to identify PDZ domain proteins associated to a given protein of interest. As proof of example, we focus our
attention on TANC1 which is a scaffold protein harboring a PDZBM at its carboxy-terminus. Using these
two approaches, we identified several PDZ domain containing proteins. Some of them were found with
both approaches, and some were specifically identified using peptide-based purification procedure. This
exemplifies advantages and differences of both strategies to identify PDZ interactions.
Key words PDZ, Purification, Protein complexes, Proteomics
1 Introduction
Over the last 20 years and following the seminal work of Bertrand
Se ´raphin and colleagues, proteomic methods deciphering the composition of protein complexes associated to proteins of interest have
become indispensable for determining their functions [1]. They
have benefited from the tremendous efforts made to improve the
sensitivity of mass spectrometry (MS) equipment and from the
development of versatile purification procedures such as proteinor peptide-based purification protocols [2]. These strategies of
purification are fast (less than 24 h) and allow the copurification
of low affinity associated proteins such as those mediated by PDZ
domains (Fig. 1). It is indeed well known that Postsynaptic density
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Avais M. Daulat and Ste ´phane Audebert contributed equally to this work.
17
Identification of PDZ Interactions by Affinity Purification
and Mass Spectrometry Analysis
Avais M. Daulat, Ste ´ phane Audebert, Mo ˆ nica Wagner, Luc Camoin,
and Jean-Paul Borg
Abstract
Identification of protein networks becomes indispensable for determining the function of a given protein of
interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This
motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and
maintenance of cell architecture. In the present chapter, we present two complementary approaches
(immunopurification and peptide-based purification procedures) followed by mass spectrometry analysis
to identify PDZ domain proteins associated to a given protein of interest. As proof of example, we focus our
attention on TANC1 which is a scaffold protein harboring a PDZBM at its carboxy-terminus. Using these
two approaches, we identified several PDZ domain containing proteins. Some of them were found with
both approaches, and some were specifically identified using peptide-based purification procedure. This
exemplifies advantages and differences of both strategies to identify PDZ interactions.
Key words PDZ, Purification, Protein complexes, Proteomics
1 Introduction
Over the last 20 years and following the seminal work of Bertrand
Se ´raphin and colleagues, proteomic methods deciphering the composition of protein complexes associated to proteins of interest have
become indispensable for determining their functions [1]. They
have benefited from the tremendous efforts made to improve the
sensitivity of mass spectrometry (MS) equipment and from the
development of versatile purification procedures such as proteinor peptide-based purification protocols [2]. These strategies of
purification are fast (less than 24 h) and allow the copurification
of low affinity associated proteins such as those mediated by PDZ
domains (Fig. 1). It is indeed well known that Postsynaptic density
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Avais M. Daulat and Ste ´phane Audebert contributed equally to this work.
17