title: Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? a comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry.
abstract: 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 (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N''-triacetylchitotriose (NAG3). The best KD value compared with solution data were found for ESSI, 19.4 +/- 3.6 microM. Then, we determined the KDs of the two nucleotide binding sites of adenylate kinase (AK), where we obtained KDs of 2.2 +/- 0.8 microM for the first and 19.5 +/- 8.0 microM 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 KD 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.
authors: Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.
authors: Matthias Conradin
authors: MC
authors: Jecklin
authors: David
authors: D
authors: Touboul
authors: Cédric
authors: C
authors: Bovet
authors: Arno
authors: A
authors: Wortmann
authors: Renato
authors: R
authors: Zenobi
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002645:Chickens; D006801:Humans; D008024:Ligands; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017186:Titrimetry; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D047428:Protein Kinase Inhibitors; D011506:Proteins; D014312:Trisaccharides; C019117:N,N',N''-triacetylchitotriose; D000244:Adenosine Diphosphate; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D000263:Adenylate Kinase; C421864:hen egg lysozyme; D009113:Muramidase