UV light-induced cross-linking of nucleosides, nucleotides and a dinucleotide to the carboxy-terminal heptad repeat peptide of RNA polymerase II as studied by mass spectrometry.
We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2'-deoxyuridine), two nucleotides (thymidine-5'-monophosphate and 5-bromo-2'-deoxyuridine-5'-monophosphate) and a dinucleotide (thymidylyl-[3'-->5']-2'-deoxyadenosine). The peptide picked was SPSYSPT (L-seryl-L-prolyl-L-seryl-L-tyrosyl-L-seryl-L-prolyl-L-threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.
Connor
D A
DA
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Falick
A M
AM
Shetlar
M D
MD
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D013058:Mass Spectrometry; D008970:Molecular Weight; D009696:Nucleic Acids / Q000737:chemistry* / Q000528:radiation effects*; D010455:Peptides / Q000737:chemistry* / Q000528:radiation effects*; D010777:Photochemistry; D012319:RNA Polymerase II / Q000737:chemistry / Q000528:radiation effects; D012091:Repetitive Sequences, Nucleic Acid; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009696:Nucleic Acids; D010455:Peptides; D012319:RNA Polymerase II