Linked Life Data

17349801

Source:http://linkedlifedata.com/resource/pubmed/id/17349801

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2007-4-27
pubmed:abstractText
Protein-RNA interactions within ribonucleoprotein particles (RNPs) can be investigated by UV-induced crosslinking of proteins to their cognate RNAs and subsequent isolation and mass-spectrometric analysis of crosslinked peptide-RNA oligonucleotides. Because of the low crosslinking yield, a major challenge in protein-RNA UV crosslinking is the detection of the crosslinked species over the excess of non-crosslinked material, especially when complex systems (native RNPs) are investigated. Here, we applied a novel approach that uses on-line nanoLC-ESI-MS/MS to detect and subsequently sequence peptide-RNA oligonucleotide crosslinks from crude mixtures. To detect the crosslinks we made use of features shared by crosslinks and phosphopeptides, that is, the phosphate groups that both carry. A precursor ion scan for m/z 79 (negative-ion mode, -ve) is applied to selectively detect analytes bearing the phosphate-containing species (i.e., residual non-crosslinked RNA and peptide-RNA crosslinks) from crude mixtures and to determine their exact m/z values. On this basis, a multiple reaction monitoring (MRM) experiment monitors the expected decomposition from the different precursor charge states of the putative crosslinks to one of the four possible RNA nucleobases [m/z 112, 113, 136, 152 (positive-ion mode, +ve)]. On detection, a high-quality MS/MS is triggered to establish the structure of the crosslink. In a feasibility study, we detected and subsequently sequenced peptide-RNA crosslinks obtained by UV-irradiation of (1) native U1 snRNPs and (2) [15.5K-61K-U4atac] snRNPs prepared by reconstitution in vitro. MRM-triggered collision-induced dissociation (CID) MS/MS enabled us to obtain sequence information about the crosslinked peptide and RNA moiety.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
1044-0305
pubmed:author
pubmed:issnType
Print
pubmed:volume
18
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
869-81
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Detection of protein-RNA crosslinks by NanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments.
pubmed:affiliation
Applera Deutschland GmbH, Darmstadt, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't