Direct observation of UV‐crosslinked protein–nucleic acid complexes by matrix‐assisted laser desorption lonization mass spectrometry

Abstract

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.

Identification and Sequence Analysis of Contact Sites between Ribosomal Proteins and rRNA in Escherichia coli 30 S Subunits by a New Approach Using Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Combined with N-terminal Microsequencing

“…To determine and sequence the oligoribonucleotides crosslinked to peptides in 30 S ribosomal subunits, we have successfully applied MALDI-MS. To date, similar studies using MALDI-MS have only been carried out on cross-linked protein-DNA complexes (36), but without oligonucleotide sequence determination.…”

Detection of protein-RNA crosslinks by nanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments

“…In pioneering studies, Jensen et al [28] and Steen et al [29] demonstrated the mass spectrometric analysis of peptide-DNA oligonucleotides by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), respectively. Other studies of protein-nucleic acid crosslinks using mass spectrometry followed [9, 10, 30 -33].…”

Identification of Specific Carboxyl Groups on Uracil-DNA Glycosylase Inhibitor Protein That Are Required for Activity

“…Mass Spectrometry-MALDI mass spectrometry was performed by the Mass Spectrometry Facilities and Service Core Unit (Environmental Health Science Center, Oregon State University) using a custombuilt time of flight instrument (31). Two different matrices were used in the sample preparation: (i) 10 mg/ml of sinapinic acid dissolved in 33% acetonitrile and 67% trifluoroacetic acid (0.1% solution) was mixed (3:1) with samples (0.5 l) containing 10 M modified or unmodified Ugi protein; and (ii) a saturated solution of ␣-cyano-4-hydroxycinnamic acid in 33% acetonitrile and 67% trifluoroacetic acid (0.1% solution) was mixed (10:1) with samples (0.5 l) containing CNBr-treated Ugi, purified peptide C3, and Asp-N-digested Ugi protein.…”