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Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.

ABSTRACT: Protein-RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto non-characterized protein-RNA contacts in ribonucleoprotein particles, as, for example, within spliceosomes. Here, we describe an improved methodology for the sequence analysis of purified peptide-RNA oligonucleotide cross-links that is based solely on MALDI-ToF mass spectrometry. The utility of this methodology is demonstrated on cross-links isolated from UV-irradiated spliceosomal particles; these were (1) [15.5K-61 K-U4 atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro, and (2) U1 snRNP particles purified from HeLa cells. We show that the use of 2',4',6'-trihydroxyacetophenone (THAP) as MALDI matrix allows analysis of cross-linked peptide-RNA oligonucleotides in the reflectron mode at high resolution, enabling sufficient accuracy to assign unambiguously cross-linked RNA sequences. Most important, post-source decay (PSD) analysis under these conditions was successfully applied to obtain sequence information about the cross-linked peptide and RNA moieties within a single spectrum, including the identification of the actual cross-linking site. Thus, in U4 atac snRNA we identified His 270 in the spliceosomal U4/U6 snRNP-specific protein 61 K (hPrp31p) cross-linked to U 44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U 30 of U1 snRNA. This type of analysis is applicable to any type of RNP complex and may be expected to pave the way for the further analysis of protein-RNA complexes in much lower abundance and/or of cross-links that are obtained in low yield.

SUBMITTER: Kuhn-Holsken E

PROVIDER: S-EPMC1370879 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.

Kühn-Hölsken Eva E Lenz Christof C Sander Björn B Lührmann Reinhard R Urlaub Henning H

RNA (New York, N.Y.) 20051201 12

Protein-RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto non-characterized protein-RNA contacts in ribonucleoprotein particles, as, for example, within spliceosomes. Here, we describe an improved methodology for the sequence analysis of purified peptide-RNA oligonucleotide cross-links that is based solely on MALDI-ToF mass spectrometry. The utility of this methodology is demonstrated on cross-links isolated from UV-irradiated spliceosomal particles; thes ...[more]

PMID: 16314460

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Project description:BackgroundIn the last decade, an innovative approach has emerged for arthropod identification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Increasing interest in applying the original technique for arthropod identification has led to the development of a variety of procedures for sample preparation and selection of body parts, among others. However, the absence of a consensual strategy hampers direct inter-study comparisons. Moreover, these different procedures are confusing to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS is therefore a necessity, and would notably enable the sharing of reference MS databases. Here, we assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling.MethodsThree homogenization methods, two of which were manual and one automatic, were used on three distinct body parts (legs, thorax, head) of two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti, and the results evaluated. The reproducibility of MS profiles, identification rate with relevant scores and the suitability of procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood-feeding and geographical origin were evaluated using both laboratory strains and field-collected mosquitoes.ResultsRelevant score values for mosquito identification were obtained for all the three body parts assayed using MALDI-TOF MS profiling; however, the thorax and legs were the most suitable specimens, independently of homogenization method or species. Although the manual homogenization methods were associated with a high rate of identification on the three body parts, this homogenization mode is not adaptable to the processing of a large number of samples. Therefore, the automatic homogenization procedure was selected as the reference homogenization method. Blood-feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from original blood in the MS profiles of the three body parts tested from both species. Finally, a significant improvement in identification scores was obtained for field-collected specimens when MS spectra of species from the same geographical area were added to the database.ConclusionThe results of the current study establish guidelines for the selection of mosquito anatomic parts and modality of sample preparation (e.g. homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database.

Dataset Information

Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.

Publications

Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.

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