In line desalting mass spectrometry for the study of noncovalent biological complexes.
Electrospray ionization-mass spectrometry is becoming widely used as a high-throughput method for the study of biomolecular interactions. It allows for the analysis of complexes from heterogeneous mixtures with high sensitivity and selectivity. In many cases, biomolecules and their complexes must be stored in nonvolatile salt buffers and other solubilizing agents, such as organics or detergents, to maintain stability and integrity. To ensure an efficient electrospray process, desalting and exchanging the biomolecular solutions into a volatile buffer is imperative. Current off-line or on-line methods to accomplish this are time-consuming, frequently disrupt noncovalent interactions, and can result in considerable sample loss. Here we describe a simple, general, and highly efficient, rapid in-line desalting approach using a small gel cartridge to assist in the mass spectrometric analysis of biomolecules and their complexes. Though the method has broad applicability, we focus our analysis on proteins and demonstrate its usefulness by examining protein-metal, protein-protein, protein-DNA, and protein-RNA interactions. The method is shown to provide rapid direct analysis of analyte solutions containing salts, glycerol, organics, and involatile buffers without deleterious effects.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA. john_cavanagh@ncsu.edu
John
J
Cavanagh
Linda M
LM
Benson
Richele
R
Thompson
Stephen
S
Naylor
D001704:Biopolymers / Q000737:chemistry*; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry; D007202:Indicators and Reagents; D009841:Oligonucleotides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001704:Biopolymers; D007202:Indicators and Reagents; D009841:Oligonucleotides; D011506:Proteins; D012313:RNA; D004247:DNA