A new functional, chemical proteomics technology to identify purine nucleotide binding sites in complex proteomes.
Adenine nucleotides are small, abundant molecules that bind numerous proteins involved in pivotal cellular processes. These nucleotides are co-factors or substrates for enzymes, regulators of protein function, or structural binding motifs. The identification of nucleotide-binding sites on a proteome-wide scale is tempting in view of the high number of nucleotide-binding proteins, their large in vivo concentration differences, and the various functions they exert. Here, we report on a functional, chemical, gel-free proteomics technology that allows the identification of protein adenine nucleotide-binding site(s) in cell lysates. Our technology uses a synthetic ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), as an affinity/activity-based probe for nucleotide-binding sites. When applied on a cellular level, 185 different FSBA-labeled sites in a human Jurkat cell lysate were identified. Functional and structural aspects of the use of FSBA on a proteome-wide scale are discussed.
Hanoulle
Xavier
X
Department of Medical Protein Research and Biochemistry, Flanders Interuniversity Institute for Biotechnology and Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium.
Van Damme
Jozef
J
Staes
An
A
Martens
Lennart
L
Goethals
Marc
M
Vandekerckhove
Joël
J
Gevaert
Kris
K
D000225:Adenine / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives; D001665:Binding Sites / Q000235:genetics; D002457:Cell Extracts / Q000737:chemistry; D002853:Chromatography, Liquid; D006801:Humans; D019169:Jurkat Cells; D008958:Models, Molecular; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
D002457:Cell Extracts; D011506:Proteins; D020543:Proteome; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000225:Adenine; D000241:Adenosine