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 noncharacterized 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-61K-U4atac] 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 0 ,4 0 ,6 0 -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 U4atac snRNA we identified His270 in the spliceosomal U4/U6 snRNP-specific protein 61K (hPrp31p) cross-linked to U44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U30 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.

RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins

“…This is the more surprising because one of the two UV cross-linking sites of hPrp31 on U4 snRNA maps to this region (14). Furthermore, in the U4atac snRNA penta-loop, which has a different sequence, similar cross-links are observed to hPrp31 (18), indicating that various contacts can be detected by UV cross-linking, depending on which base is in contact with the protein surface. Since this RNA-protein interaction pattern is not required for primary recognition in the ternary complex, it is presumably important in one of the dynamic functional states of U4 snRNA during the splicing cycle.…”

“…By ultraviolet irradiation of the U4atac-snRNP and the native purified U4/U6⅐U5 tri-snRNP particle, hPrp31 has been crosslinked to single-stranded nucleotide regions at the three-way junction of the base-paired U4 and U6 snRNAs and the terminal penta-loop of the U4atac and the U4 snRNAs (14,18). This argues for the existence of direct contacts between hPrp31 and the U4 snRNA.…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…The cross-linked nucleotide is determined by calculating the mass difference between the entire mass of the cross-linked species and the mass of the cross-linked peptide. In addition, marker ions of the cross-linked nucleotides in the lower [37, 43,44]. Currently, electrospray ionization (ESI) MS coupled to a nano-liquid chromatography (LC) is the method of choice for analyzing such cross-links.…”

Collision induced dissociation-based characterization of nucleotide peptides: Fragmentation patterns of microcin C7–C51, an antimicrobial peptide produced by Escherichia coli

“…If the complex life time is sufficiently large, due to the relative low internal energy compared to the dissociation energy threshold, internal proton transfer can occur, resulting from the formation of complementary ion pairs. Note that such isomerizations at the first step of the fragmentation pathways, yielding ionneutral complexes, can be generalized to rationalize the fragmentation patterns of certain protein-RNA cross-links analyzed by post-source decay (PSD) experiments [3,4]. [29,30].…”

“…This indicates that these species encompass the peptide part and result from peptide backbone cleavage. To characterize the structure of the m/z 445 and 630 product ions, pseudo-MS 3 and MS 3 experiments were performed on both the Qq-TOF and the LTQ Orbitrap instruments. Fragmentation of the selected ion at m/z 630 triggered the formation of the m/z 445 ion exclusively.…”