Protein-DNA arrays as tools for detection of protein-protein interactions by mass spectrometry
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Protein-DNA arrays as tools for detection of protein-protein interactions by mass spectrometry. / Gogolin, Lars; Schroeder, Hendrik; Itzen, Aymelt; Goody, Roger S; Niemeyer, Christof M; Becker, Christian F W.
in: CHEMBIOCHEM, Jahrgang 14, Nr. 1, 02.01.2013, S. 92-9.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Protein-DNA arrays as tools for detection of protein-protein interactions by mass spectrometry
AU - Gogolin, Lars
AU - Schroeder, Hendrik
AU - Itzen, Aymelt
AU - Goody, Roger S
AU - Niemeyer, Christof M
AU - Becker, Christian F W
N1 - Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2013/1/2
Y1 - 2013/1/2
N2 - Analysis of multiple protein-protein interactions using microarray technology remains challenging, and site-specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein-DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt-DNA hybrid molecules described here are used for DNA-directed immobilization on glass- and silica-based microarrays. Methods for the detection of protein-DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP- and GTP-bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt-effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.
AB - Analysis of multiple protein-protein interactions using microarray technology remains challenging, and site-specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein-DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt-DNA hybrid molecules described here are used for DNA-directed immobilization on glass- and silica-based microarrays. Methods for the detection of protein-DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP- and GTP-bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt-effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.
KW - Base Sequence
KW - DNA
KW - Humans
KW - Immobilized Proteins
KW - Mass Spectrometry
KW - Nucleic Acid Hybridization
KW - Oligonucleotide Array Sequence Analysis
KW - Protein Array Analysis
KW - Protein Interaction Mapping
KW - ras Proteins
KW - Journal Article
U2 - 10.1002/cbic.201200597
DO - 10.1002/cbic.201200597
M3 - SCORING: Journal article
C2 - 23208955
VL - 14
SP - 92
EP - 99
JO - CHEMBIOCHEM
JF - CHEMBIOCHEM
SN - 1439-4227
IS - 1
ER -