Biología y Biomedicina >
Centro de Investigaciones Biológicas (CIB) >
(CIB) Artículos >
Closed Access item Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples
|Authors:||Ribeiro, João P.|
Cañada Vicinay, Francisco Javier
Naranjo, José R.
|Keywords:||1D diffusion-ordered spectroscopy (DOSY), Nuclear magnetic resonance (NMR), Small-molecule contaminants, Imidazoles, Diffusion, Protein purification, Lectin|
|Citation:||Analytical Biochemistry 396(1):117-123(2010)|
|Abstract:||Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers.|
|Description:||Supplementary data can be obtained at: http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S0003269709006071/1-s2.0-S0003269709006071-mmc1.doc/272363/html/S0003269709006071/4a58356491e3b2153684edba66691f9a/mmc1.doc|
|Publisher version (URL):||http://dx.doi.org/10.1016/j.ab.2009.09.001|
|Appears in Collections:||(CIB) Artículos|
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.