Direct observation of UV-crosslinked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry.
Interactions between proteins and nucleic acids are important in the fundamental cellular processes that drive replication, recombination, dynamic alteration and repair of DNA, transcription and processing of RNA, synthesis of proteins, and regulation of enzyme activities. As part of an effort to develop a general, sensitive mass spectrometric strategy for the characterization of protein-nucleic acid interactions, we have used matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to analyze protein-nucleic acid complexes that have been covalently crosslinked by ultraviolet (UV) light. In general, the application of MALDI mass spectrometric techniques to studies of UV-induced crosslinking of nucleoprotein complexes is demonstrated to be feasible. Specifically, MALDI mass analysis was used to determine the molecular weights of the phage T4 gene 32 protein (gp32) crosslinked to the oligonucleotide (dT)20, and the Escherichia coli transcription termination factor rho, photoaffinity labeled with 4-thio-uridine-diphosphate (4sUDP). The covalent gp32:(dT)20 complex is readily detected at a concentration of 1-2 microM in 1 microL of an unpurified solution of reactants that has been exposed to a single, 266 nm UV laser pulse. Mass spectrometric molecular weight determinations of the covalent rho:4sUDP complex add directness and specificity to the ATPase inactivation assay normally used to monitor the formation of 4sUDP photoaffinity labeled rho. It is found that successful MALDI mass spectrometry of protein-nucleic acid complexes is as critically dependent on the choice of solvents and additives as it is on the primary matrix compound.
Jensen
O N
ON
Department of Biochemistry & Biophysics, Oregon State University, Corvallis 97331-7305.
Barofsky
D F
DF
Young
M C
MC
von Hippel
P H
PH
Swenson
S
S
Seifried
S E
SE
D013058:Mass Spectrometry / Q000379:methods*; D009696:Nucleic Acids / Q000528:radiation effects*; D011506:Proteins / Q000528:radiation effects*; D014466:Ultraviolet Rays
D009696:Nucleic Acids; D011506:Proteins