Detection of ATP-binding to growth factors.
It was shown in previous work that the interaction of growth factors (GFs) with adenosine triphosphate (ATP) is essential for their neuroprotective effect. Here we investigated the nature of the association of human basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) with ATP. It was demonstrated that this interaction involves the formation of non-covalent ATP-GF complexes that are labile at low pH and that could be selectively purified and subjected to electrospray and MALDI-TOF mass spectrometry. The results obtained with these techniques indicated that the stability of the complexes is high. Main features of the procedure used here are: (1) reversed-phase purification of nucleotide-protein non-covalent complexes, (2) their detection with MALDI-TOF-MS using acid-free matrix, and/or (3) their measurement with ESI-MS using soft desolvation conditions. The methodology was successful in providing proof for the presence of various nucleotide-GF complexes. It was extended to other nucleotide-binding proteins (ribonuclease A) as well as proteins that do not exhibit nucleotide binding (lysozyme) as positive and negative control, respectively. Thus, the method demonstrated its general use for the investigation of a wide range of proteins interacting with nucleotides as long as their complexes are sufficiently stable to accommodate the experimental conditions.
Integrated Functional Genomics, Interdisciplinary Center of Clinical Research, University of Münster, Münster, Germany. koenigs@uni-muenster.de
Simone
S
König
Anja
A
Hasche
Stefanie
S
Pallast
Josef
J
Krieglstein
Susanne
S
Klumpp
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019208:Brain-Derived Neurotrophic Factor / Q000737:chemistry / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020932:Nerve Growth Factor / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D019208:Brain-Derived Neurotrophic Factor; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D000255:Adenosine Triphosphate; D020932:Nerve Growth Factor