期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 19, 期 3, 页码 332-343出版社
SPRINGER
DOI: 10.1016/j.jasms.2007.11.007
关键词
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We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (K,) of the model system hen egg white lysozyme (HEWL) binding to N,N',N ''-triacetylchitotriose (NAG(3)). The best K-D value compared with solution data were found for ESSI, 19.4 +/- 3.6 mu M. Then, we determined the K(D)s of the two nucleotide binding sites of adenylate kinase (AK), where we obtained K(D)s of 2.2 +/- 0.8 mu M for the first and 19.5 +/- 8.0 mu M for the second binding site using ESSI. We found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the K-D for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found KD values with a strong charge state dependence, which were in no case close to literature values for solution phase.
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