Protein-DNA arrays as tools for detection of protein-protein interactions by mass spectrometry

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 -