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Detection of Protein–RNA Crosslinksby NanoLC-ESI-MS/MS Using PrecursorIon Scanning and Multiple ReactionMonitoring (MRM) Experiments

Christof LenzApplera Deutschland GmbH, Darmstadt, Germany

Eva Kühn-Hölsken and Henning UrlaubBioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen,Germany

Protein–RNA interactions within ribonucleoprotein particles (RNPs) can be investigated byUV-induced crosslinking of proteins to their cognate RNAs and subsequent isolation andmass-spectrometric analysis of crosslinked peptide–RNA oligonucleotides. Because of the lowcrosslinking yield, a major challenge in protein–RNA UV crosslinking is the detection of thecrosslinked species over the excess of non-crosslinked material, especially when complexsystems (native RNPs) are investigated. Here, we applied a novel approach that uses on-linenanoLC-ESI-MS/MS to detect and subsequently sequence peptide–RNA oligonucleotidecrosslinks from crude mixtures. To detect the crosslinks we made use of features shared bycrosslinks and phosphopeptides, that is, the phosphate groups that both carry. A precursor ionscan for m/z 79 (negative-ion mode, �ve) is applied to selectively detect analytes bearing thephosphate-containing species (i.e., residual non-crosslinked RNA and peptide–RNAcrosslinks) from crude mixtures and to determine their exact m/z values. On this basis, amultiple reaction monitoring (MRM) experiment monitors the expected decomposition fromthe different precursor charge states of the putative crosslinks to one of the four possible RNAnucleobases [m/z 112, 113, 136, 152 (positive-ion mode, �ve)]. On detection, a high-qualityMS/MS is triggered to establish the structure of the crosslink. In a feasibility study, wedetected 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 sequenceinformation about the crosslinked peptide and RNA moiety. (J Am Soc Mass Spectrom 2007,18, 869–881) © 2007 American Society for Mass Spectrometry

RNA and protein–RNA complexes (ribonucleo-protein particles, RNPs) are just as crucial in thecontrol and regulation of gene expression in the

eukaryotic cell as are protein–DNA complexes (tran-scriptomes). Very recent investigations, such as in thefield of alternative splicing regulation and of transla-tional control by microRNAs (miRNA), have demon-strated impressively that protein–RNA interactions andtheir dynamic changes are the complex driving forcesbehind these processes (for review, see Blencowe [1]and Bartel [2]).Numerous MS-based proteomic approaches deter-

mined which proteins are associated with RNA [3–7].However, these studies have yielded only limited infor-

mation about which protein is in direct contact with theRNA. A straightforward method to determine this iscrosslinking.Crosslinking between RNA and proteins can be

achieved in different ways: (1) by incorporating baseanalogues (e.g., 5-bromo-2=-deoxyuridine [8], iodo-derivatives [9, 10], or 4-thio-uracil [11–13]) into theRNA, either site-specifically or randomly; (2) by chem-ical modification of the RNA backbone (acidophenacyland benzophenone [14, 15]) (3) by incubating protein–RNA assemblies with crosslinking reagents (e.g., form-aldehyde [16], methylene blue [17], and cis-platin [18]);or (4) by direct UV-irradiation at 254-nm wavelength[19]. The latter method uses the naturally occurring UVreactivity of the RNA nucleobases and has been suc-cessfully applied to various native protein–RNA com-plexes isolated from living cells [20–22].Once the crosslink has been established, the main

challenge is the identification of the crosslinked protein.

Published online March 8, 2007Address reprint requests to: Dr. Henning Urlaub, Bioanalytical MassSpectrometry Group, Max Planck Institute for Biophysical Chemistry, AmFassberg 11, 37077 Göttingen, Germany. E-mail: [email protected]

© 2007 American Society for Mass Spectrometry. Published by Elsevier Inc. Received November 17, 20061044-0305/07/$32.00 Revised January 18, 2007doi:10.1016/j.jasms.2007.01.013 Accepted January 22, 2007

This can be achieved by the use of highly specific androbust methods, such as strongly binding antibodies.Immunoprecipitation of crosslinked protein–nucleicacid complexes has been successfully applied to theanalysis of the interacting nucleotide parts and is man-ifested in generally applicable approaches like chroma-tin immunoprecipitation (ChIP) [23, 24], crosslinkingand immunoprecipitation (CLIP) [25], and/or (chroma-tin) immunoprecipitation followed by DNA microarrayanalysis [(Ch)IP-chip] [26, 27]. However, these ap-proaches presuppose knowledge of an interacting pro-tein or factor and are thus hypothesis-driven. Conse-quently, the identification of hitherto unknowncrosslinked proteins requires other protein-analyticalmethods, of which mass spectrometry is the mostsensitive technique currently available.Protein–nucleic acid crosslinks have been investi-

gated by mass spectrometry in several laboratories. Inpioneering studies, Jensen et al. [28] and Steen et al. [29]demonstrated the mass spectrometric analysis of peptide–DNA oligonucleotides by matrix-assisted laser desorp-tion/ionization (MALDI) and electrospray ionization(ESI), respectively. Other studies of protein–nucleic acidcrosslinks using mass spectrometry followed [9, 10,30–33].We are particularly interested in the characterization

of peptide–RNA conjugates from native protein–RNAcomplexes isolated from a living cell. This demandshighly sensitive methods.We therefore established in previous studies a puri-

fication strategy that enabled us to isolate crosslinkedpeptide–RNA oligonucleotides from native and recon-stituted ribonucleoprotein particles after UV-irradiation([22] and below). Our approach consists of hydrolysis ofthe protein and RNA moieties (with endoproteinasesand endonucleases, respectively) and purification of thecrosslinked peptide–RNA oligonucleotides by micro-bore and/or capillary reversed-phase (RP) liquid chro-matography (LC). Thereafter, MALDI–MS and –MS/MS(tandem mass spectrometric) analysis of the isolatedcrosslinks provide structural information about thecrosslinked peptide and RNA moieties.Because the crosslinking yield is very low, especially

when the natural UV reactivity of the RNA nucleobasesis used, a successful mass spectrometric analysis ofcrosslinked proteins must meet challenges similar tothose encountered in the detection of post-translationalmodifications: (1) Enrichment of the crosslinked (mod-ified) species over the excess of non-crosslinked (non-modified) material, (2) identification of the crosslinked(modified) species and exclusion of false positives, and(3) the application of mass spectrometric analysis per se.To detect and sequence peptide–RNA oligonucleo-

tide heteroconjugates derived from UV-irradiated RNPparticles, we make use of features shared by protein–RNA crosslinks and phosphopeptides. Both speciescarry phosphate groups, a feature that can be used todetect them in crude protein/peptide mixtures. Mucheffort is currently being devoted to enrichment strate-

gies for phosphopeptides by affinity chromatography(for review, see Jensen [34] and Corthals et al. [35]).Alternatively, experiments using mass spectrometryalone (i.e., constant neutral loss, precursor ion scan, andmultiple reaction monitoring) have also been success-fully performed to selectively detect and subsequentlysequence phosphopeptides [36–39]. In particular, mul-tiple reaction monitoring (MRM)–driven LC/MS/MSexperiments on hybrid triple quadrupole/linear ion-trap instruments are highly sensitive techniques thatallow one to identify modified components of lowabundance in complex mixtures. Although MRM is atargeted technique, it can nonetheless be applied for thedetection of hitherto unknown compounds if a suffi-cient amount of starting information is available to forma hypothesis about a set of putative analytes. Cox et al.[40] and Unwin et al. [41] demonstrated the detection oflow-abundance phosphorylation events using only theamino acid sequence of the protein concerned as astarting point.In this study, we set up a strategy for crosslink

analysis based on nanoLC-ESI-MS/MS using precursorion scanning and MRM experiments. In a feasibilitystudy, we successfully detected and identified severalpeptide–RNA oligonucleotide crosslinks derived from(1) native U1 small nuclear ribonucleoprotein (snRNP)particles [42] and (2) [15.5K-61K-U4atac snRNA] com-plexes [43]. These U snRNPs are subcomplexes of thehuman major and minor spliceosome, respectively;each of these spliceosomes is a large protein–RNAmachinery that catalyzes removal of the introns frompre-mRNAs and subsequent ligation of the exons toyield mature mRNAs (for review, see Will and Lühr-mann [44]). U1 snRNP particles consist of a smalluridine-rich RNA (U1 snRNA) and ten proteins (70K, Aand C protein and the seven Sm-proteins B/B=, D1, D2,D3, E, F, and G), whereas the [15.5K-61K-U4atacsnRNA] complexes reconstituted in vitro consist of theU4atac snRNA and the evolutionarily conserved pro-teins 15.5K [45] and 61K [46].

Experimental

Sample Preparation, Crosslinking, and Purificationof Peptide–RNA Oligonucleotide Crosslinks

Native U1 snRNP and [15.5K-61K-U4atac snRNA] par-ticles generated by reconstitution in vitro were pre-pared as described previously [42, 43]. UV crosslinkingof samples was carried out at 254 nm for 2 minaccording to Kühn-Hölsken et al. [22]. A°total°of°50pmol of crosslinked sample was denatured in the pres-ence of 6 M guanidine hydrochloride (guanidine-HCl)in 50 mM Tris-(hydroxymethyl)aminomethane (Tris–HCl) (pH 8.0), heated for 5 min at 90 °C, cooled to roomtemperature, diluted with 50 mM Tris-HCl (pH 8.0) to 1M guanidine-HCl, and incubated overnight at 37 °Cwith trypsin or chymotrypsin at an enzyme-to-substrateratio of 1:20. Trypsin and chymotrypsin were tested for

870 LENZ ET AL. J Am Soc Mass Spectrom 2007, 18, 869–881

nuclease activity before use. Samples were precipitatedwith 3 volumes of ethanol and 1/10 volume of 3.0 Msodium acetate pH 5.3; dissolved in a buffer containing150 mM sodium chloride, 50 mM Tris–HCl (pH 7.5), 1mM ethylendiamine tetraacetate (EDTA), and 0.1% so-dium dodecylsulfate (SDS); and loaded onto a Seph-adex 75HR size-exclusion column (GE Healthcare, 32 �0.5 cm) mounted in a SMART system (GE Healthcare).Fractions containing RNA were precipitated with etha-nol as above and dissolved in a buffer containing 50mM Tris-HCl (pH 7.5) and 1 mM EDTA. RNA washydrolyzed with 1–2 �g RNase A and/or T1 (Ambion)for 2 h at 52 °C. Residual longer peptides were againhydrolyzed with 1 �g of the respective endoproteinasesat 37 °C overnight. Aliquots (10–20 �L) of the crudecrosslinked mixture were stored at �20 °C for nanoLCseparation.

NanoLC/MS/MS Analysis of Mixtures ContainingCrosslinks

Mixtures containing peptide–RNA oligonucleotidecrosslinks were separated on a Tempo 1D nanoLCsystem (Applied Biosystems/MDS Sciex). Ten percentof the preparation to be analyzed (nominal 5 pmolwhen referring to the amount of starting material usedfor the experiments) was injected onto a PepMap RP-C18 trap column (5 � 300 �m, 5 Å, Dionex), purified,and desalted with an extended washing time to reducethe content of pure RNA compounds [0.5% (vol/vol)formic acid (FA)/2% (vol/vol) CH3CN, 20 �L/min, 8min). Separation was achieved by back-flush gradientelution onto a PepMap C18 microcolumn [15 cm � 75�m, 3 Å; 90-min linear gradient 5–40% (vol/vol)CH3CN/0.1% (vol/vol) FA, 300 nL/min]. The eluentwas analyzed in a 4000 Q TRAP LC/MS/MS systemusing a Microionspray source and a nano-desolvation

chamber interface (nanoDCI). Analyst 1.4.1 and Bioana-lyst 1.4.1 software (all Applied Biosystems/MDS Sciex)were used for acquisition control, information-dependent experiments, and data processing.

Precursor Ion Scanning Experiments

Peptide–RNA oligonucleotide crosslinks and RNAoligonucleotides were selectively detected using aprecursor ion scan experiment for m/z 79 (PO3

�). The400–1800 m/z range was scanned using a scan time of5 s and a rolling collision energy of (0.04 m/z � 9) V.The most abundant signals detected in the precursorion scan (up to five signals) were then submitted to ahigh-resolution linear ion-trap MS scan in negative-ion mode to determine unambiguously their chargestate and molecular weight (MW). The longest cycletime was 6 s. The linear ion-trap scans were thencharge-state deconvoluted to yield a “molecularweight versus retention time” map (“heat map”) ofthe putative crosslinks in each sample.

Multiple Reaction Monitoring Experiments

Fragmentation reactions were predicted based on (1)each putative crosslink being present as either[M�2H]2� or [M�3H]3�; (2) a Q2 collision energy of 65and 100 V for triply and doubly charged precursors,respectively; and (3) the generation of RNA nucleobase-specific fragments m/z 112 (C), 113 (U), 136 (A), and 152(G). MRM transitions were set up in positive-ion mode(�ve) for each of these fragmentations and monitoredthroughout the gradient using unit resolution in Q1 andQ3,°and°dwell°times°of°30°–°60°ms°per°transition.°Table°1shows an example of the design of MRM transitions.Two doubly charged precursors were detected in thenegative-mode precursor ion scanning experiment for

Table 1. Example of the calculation of MRM transitions from precursors detected in the precursor ion scanning experiment of UV-irradiated native U1 snRNP particles after hydrolysis with trypsin and RNase T1

Precursor ion scan m/z 79 MRM to RNA nucleobase

[M-2H]2� RT (min) MW (Da) m/z Q1 CE (V) m/z Q3

961.4 25.0 1924.8 642.6 (3�) 65 112.0 (C)642.6 (3�) 65 113.0 (U)642.6 (3�) 65 136.0 (A)642.6 (3�) 65 152.0 (G)963.4 (2�) 100 112.0 (C)963.4 (2�) 100 113.0 (U)963.4 (2�) 100 136.0 (A)963.4 (2�) 100 152.0 (G)

883.3 31.0 1768.7 590.5 (3�) 65 112.0 (C)590.5 (3�) 65 113.0 (U)590.5 (3�) 65 136.0 (A)590.5 (3�) 65 152.0 (G)885.3 (2�) 100 112.0 (C)885.3 (2�) 100 113.0 (U)885.3 (2�) 100 136.0 (A)885.3 (2�) 100 152.0 (G)

871J Am Soc Mass Spectrom 2007, 18, 869–881 LC-ESI-MS/MS Analysis of Protein–RNA Crosslinks

the native U1 snRNP particles, m/z 961.4 and m/z 883.3.These two precursors were translated into a panel of 16MRM transitions, accounting for doubly/triply chargedprecursors and the four expected nucleobase markerfragments in the positive-ion mode MRM experiment.On exceeding a threshold value on one of the MRM

transitions, up to two high-resolution MS and up to twofull product ion scans in positive-ion mode were trig-gered. A dynamic subtraction of the MRM backgroundwas used, to ensure that MS/MS was performed close tothe apex of the chromatographic profile for each precur-sor. Product ion scans were performed by using isolationof the precursor in Q1 at a resolution of �1.0 FWHM, Q2collision-cell activation using rolling collision energy andnitrogen as collision gas, and recording of the fragments inthe Q3 linear ion trap. Product ion spectra were charge-state deconvoluted to allow interpretation.

Results and Discussion

Overall Strategy for ESI-MS to Detectand Sequence Peptide–RNA OligonucleotideCrosslinks from a Crude Mixture

In our previous studies, the successful MS analysis ofpeptide–RNA crosslinks was mainly dependent on thedetection of the crosslinks in the corresponding RP-HPLC fraction by monitoring the absorbance at 220 nm(peptide) and 260 nm (RNA). However, performing thistype of detection before MS suffers from a certainweakness, such as: residual non-crosslinked peptideswithin the fractions interfere with, and sometimes pre-vent, the detection of the crosslinked species in theMALDI spectrum, especially when only small amountsof crosslinked material are being investigated.To overcome this weakness in the detection of

crosslinks derived from native UV-irradiated particles,we combined, for the first time, on-line nanoLC withdirect detection and subsequent sequencing ofcrosslinks in an ESI hybrid triple quadrupole/linearion-trap (LIT) mass spectrometer. Coupling nanoLCdirectly to the mass spectrometer reduces the amount ofstarting material needed by a factor of 20 comparedwith capillary LC.The overall strategy we used for our experiments is

outlined°in°Figure°1.°A°crude°mixture°of°peptide–RNAoligonucleotide crosslinks and non-crosslinked RNAoligonucleotides derived from (1) native U1 snRNPparticles and (2) [15.5K-61K-U4atac snRNA] complexesreconstituted in vitro was prepared according to Kühn-Hölsken° et° al.° [22].° The°mixture° is° injected° onto° ananoLC system equipped with a 300-�m ID trappingcolumn and a 75-�m ID separation column of the samereversed-phase°C18°material°(Figure°1a).Peptide–RNA oligonucleotide crosslinks are retained

on the trapping column, whereas the non-crosslinkedRNA oligonucleotides do not bind during an extendedwashing period. Bound crosslinks are eluted by back-flush onto the analytical column and are separated by

using a linear organic gradient. In contrast to ourprevious studies, the on-line coupling uses �5 pmol ofstarting material—that is, about 1/20th of the amountof starting material that had been needed for crosslinkanalysis°in°previous°studies°[21,°22].The subsequent mass-spectrometric strategy on a

hybrid triple quadrupole/linear ion trap for targetingthe structural features of peptide–RNA crosslinks in acrude°mixture° is° outlined° in° Figure° 1b° and° c.° Thestrategy consists of a two-run LC/MS/MS workflow. Ina°first°nanoLC/MS/MS°run,°a°precursor°ion°scan°[36]for m/z 79 (negative-ion mode, �ve) is used to selec-tively detect analytes containing a phosphate moiety;that is, it will detect both residual non-crosslinked RNAoligonucleotides and crosslinked peptide–RNA species(Figure°1b).°Purely°peptidic°compounds,°however,°willnot be detected. A high-resolution LIT-MS (�ve) pro-vides information about accurate MW and charge state.A second nanoLC run in positive-ion mode targets

the° detected° potential° crosslinks° by° MRM° [40,° 41],monitoring their expected decomposition from differ-ent precursor charge states to one of the four possibleRNA°nucleobases°[m/z 112,°113,°136,°152;°(�ve);°Figure1c].°MRM°transitions°are°designed°assuming°both°dou-bly and triply charged precursors in Q1 because thepredominant charge state in positive-ion mode cannotbe safely assumed to be the same as in negative-ionmode. On detection, a high-quality MS/MS is triggeredto establish the structure of the crosslink. The Q2collision cell is used to activate precursors for CIDinstead of the Q3 linear ion trap. As a consequence, theresulting MS/MS spectra in most cases deliver informa-tion about both the crosslinked peptide and the RNAmoiety°of°the°analytes°(Figure°1c).Both negative- and positive-ion-mode experiments

were performed using the same acidic gradient condi-tions, with formic acid as modifier. Using constant-gradient conditions we observed retention times to bereproducible within 60 s for all of the runs in a set. Asa consequence, retention time can be used as an addi-tional firm criterion for the assignment of chromato-graphy peaks between the two polarities. This strategyalso allows rapid switching between precursor ionscanning and MRM experiments in a series of experi-ments, which is a prerequisite for routine analysis.It is currently not possible to accurately assess the

sensitivity of this approach for peptide–oligonucleotidecrosslinks because there are no purified standards avail-able in known quantities. Using synthetic phosphopep-tides of similar molecular weight, however, we haveestablished detection limits to be �5 fmol on-columnfor both negative-ion-mode precursor ion scanning andpositive-ion-mode MRM experiments with formic acidas modifier (C. Lenz, unpublished data). The sensitivityfor peptide–oligonucleotide crosslinks in this regime istherefore expected to be in the low femtomole range forboth polarities.


Figure 1. Schematic representation of the column setup (a) and the two-run LC/MS/MS workflow ona hybrid triple quadrupole/linear ion-trap mass spectrometer (b, c) used for the identification ofpeptide–RNA crosslinks derived from ribonucleoprotein particles. (a) Crude mixtures containingpeptide–RNA crosslinks and both non-crosslinked RNA oligonucleotides and peptides are loaded onto ananoLC system. While free RNA oligonucleotides are washed out, crosslinks remain on the trappingcolumn (valve pos. 1–2) and are eluted by back-flush onto an analytical column (valve pos. 1–6), wherethey are subsequently separated. (b) Precursor ion scan for m/z 79 (negative-ion mode,�ve) to selectivelydetect analytes containing a phosphate moiety. The resulting spectrum shows the precursor ion signal atthe respective retention time. A high-resolution LIT-MS (�ve) provides information about accurate MWand charge state. (c) Targeted LC/MS/MS analysis using MRM detection in positive-ion mode (�ve),monitoring the expected decomposition of potential crosslinks from different precursor charge states toone of the four possible RNA nucleobases (m/z 112, 113, 136, 152). At the respective retention times, ahigh-resolution LIT-MS and MS/MS are triggered to provide sequence information about both thecrosslinked peptide and RNA moiety of the analytes.


Precursor Ion Scan Detection, MRM,and CID-induced Fragmentation

Precursor ion scan detection. To generate peptide–RNAheteroconjugates for detection in precursor ion scan-ning experiments we (1) UV-irradiated U1 snRNP par-ticles and then treated them with endoproteinase tryp-sin and RNase T1 or RNases A and T1, and (2) UV-irradiated [15.5.K-61K-U4atac snRNA] particlesreconstituted in vitro and then treated them with chy-motrypsin and RNases A and T1. Samples were puri-fied as described above and the crude mixtures—consisting of residual non-crosslinked peptides, non-crosslinked RNA oligonucleotides, and peptide–RNAoligonucleotide crosslinks—were submitted to on-linenanoLC-ESI-MS.Figure°2°shows°representative°base-peak° intensity

chromatograms from precursor ion scan experiments(m/z 79 in �ve) of both samples. The chromatogramsreveal the elution of several analytes: putative peptide–RNA crosslinks, non-crosslinked RNA molecules,and/or (when native particles were used as startingmaterial) phosphopeptides. Treatment with RNase T1alone generally produces conjugates with a larger RNA

moiety compared with conjugates derived from treat-ment with both endonucleases because RNase T1cleaves the RNA specifically on the 3= side of guanosinenucleotides (G), whereas RNase A cleaves 3= to cytidineand uridine (C and U). Accordingly, different patternsin the LC analysis combined with precursor ion scan-ning experiments are observed. In the case of native U1snRNP°particles°digested°with°RNase°T1°(Figure°2a),°thebase-peak chromatogram does not show well-definedsignals, but instead shows relatively unspecific elutionof phosphate-containing species, with a maximum ataround 24 min that exhibits a significant amount ofstreaking. In contrast, both the native and the reconsti-tuted particles treated with RNases A and T1 showelution°profiles°with°much°sharper°peaks°(Figure°2band c) at about 26-, 13-, and 48-min retention times,respectively.The°inserts°in°Figure°2a°and°2c°show°examples°of°the

precursor ion scan signals from the samples U1 snRNPtreated°with° trypsin° and°RNase°T1° (Figure° 2a)° and[15.5.K-61K-U4atac snRNA] treated with chymotrypsinand°RNases°A°and°T1° (Figure° 2c)° at°different° timepoints of the elution, together with the corresponding

Figure 2. Base-peak chromatograms of LC/MS/MS experiments using precursor ion scan detectionform/z 79 in negative-ion mode for native U1 snRNP particles after hydrolysis with trypsin and RNaseT1 (a, b) or RNases A and T1 (b) and for [15.5K-61K-U4atac snRNA] complexes prepared byreconstitution in vitro after treatment with chymotrypsin and RNases A and T1 (c). Particles treatedwith RNases A and T1 show elution profiles with sharp peaks, whereas chromatograms of particlesderived after hydrolysis with RNase T1 alone show a relatively unspecific elution of phosphate-containing species. The inserts show the precursor ion scan signals of phosphate-containing speciesbetween m/z 400 and m/z 1800 derived from UV-irradiated U1 snRNP particles hydrolyzed withtrypsin and RNase T1 at retention time 24.5 min (a) and from [15.5.K-61K-U4atac snRNA] complexestreated with chymotrypsin and RNases A and T1 at retention time of 49.3 min (c) as well as thehigh-resolution LIT-MS scan of the signals marked by arrows to determine the charge states and themolecular weights of the respective precursor ions.


high-resolution linear ion-trap MS for determination ofthe charge state. It is important to note that under theconditions used all putative crosslinked precursorswere detected predominantly as their [M�2H]2� quasi-molecular ions in negative-ion mode.The results of the precursor ion scan experiments for

U1 snRNP particles treated with trypsin and RNase T1and for [15.5-K-61K-U4atac snRNA] complexes aftertreatment with chymotrypsin and RNases T1 and A are

plotted°in°Figure°3a°and°3b.°The°signal°intensity°of°m/z79 detection (�ve) is shown as a function of m/z and theliquid chromatography retention time. Importantly, po-tential peptide–RNA crosslinks and/or phosphopep-tides show well-defined retention times because of theirpeptide content, whereas residual non-crosslinkedRNA oligonucleotides (that are not completely re-moved from the trapping column and thus co-elutefrom the analytical column) have no defined retention

Figure 3. Results of the precursor ion scan detection for m/z 79 in negative-ion mode for U1 snRNPparticles treated with trypsin and RNase T1 (a) and [15.5.K-61K-U4atac snRNA] complexes hydro-lyzed with chymotrypsin and RNases A and T1 (b). Signal intensity ofm/z 79 detection (�ve) is shownas a function of m/z and chromatographic retention time (RT; “heat map”). Putative peptide–RNAcrosslinks and/or phosphopeptides show well-defined RTs because of their peptide content, whereasresidual non-crosslinked RNA oligonucleotides have no defined RTs and appear as streaks in the plot.Two potential crosslinks at m/z 883.4 and 961.4 could be detected in the U1 snRNP; the heat map of[15.5.K-61K-U4atac snRNA] reveals four putative crosslinks at m/z 890.3, 899.3, 1035.9, and 1045.0. Theprecursor ion signals appear as [M-2H]2� quasimolecular ions.


time and appear as streaks in the plot. Importantly, the“streaking effect” is stronger when RNase T1 is used forgeneration of oligonucleotides. This is most probably aresult of the increased length of the oligonucleotides(see above).

MRM and CID-induced fragmentation. On the basis ofthe precursor ion scan plot, we set up MRM experi-ments for those precursors that showed a definedretention time in the chromatography. The MRM exper-iments were performed in positive-ion mode. For eachprecursor the decomposition from [M�2H]2� and[M�3H]3� to each of the four RNA nucleobases ismonitored, i.e., [M�nH]n� ¡ [{111, 112, 135, 151}�H]�.As a result, eight MRM transitions are monitored foreach precursor.The predominant charge states observed in the MRM

experiments in positive-ion mode were �2 and �3,corresponding to the bimolecular nature of thecrosslinks that offers possible protonation sites both atthe peptide and RNA moieties. A dependency of thecharge state on the relative contents of the two struc-tural moieties seems likely, but more data points needto be collected to allow a reliable prediction.MRM traces of m/z 642.6 (�3) to m/z 136.1 (native U1

snRNP) and of m/z 892.3 (�2) to m/z 136.1 ([15.5.K-61K-U4atac snRNA] complexes reconstituted in vitro) areshown°in°Figure°4a°and°b,°respectively.°Q2°collision°cellproduct ion scans performed at the respective retentiontimes revealed structural information on the peptide–RNA°crosslinks°(Figures°5°and°6).°Typically,°informa-

tion about the crosslinked peptide moiety can be foundin the lower m/z range of the spectrum, whereas thehigher m/z range reveals information about thecrosslinked RNA moiety.Figure°5°shows°the°product°ion°scan°(Figure°5a)°and

the°charge-state°deconvoluted°MS/MS°spectrum°(Fig-ure°5b)° for° the°putatively° crosslinked°precursor°m/z642.6 (�3) obtained during targeted LC/MS/MS anal-ysis of native U1 snRNP particles after hydrolysis withtrypsin°and°RNase°T1.°The°insert°in°Figure°5a°shows°theyp-type fragment-ion series of the crosslinked peptidefrom yp1 to yp5, revealing the sequence VDVER, to-gether with the signals of two RNA nucleotides: c1 (A)and zr1 (C)°(see°Figure°5c°for°structural°information).The°deconvoluted°MS/MS°spectrum°in°Figure°5b°showsRNA fragments of the crosslinked nucleotides of yr-, x-,z-, and d-type, revealing the trinucleotide AUC (seeFigure°5c°for°structural°information).°The°loss°of°cyti-dine (�m � 305) and the subsequent losses of adenine(�m � 135), adenosine (�m � 329), cytosine (�m �111), and the two remaining sugar phosphates identifyuracil as the actual crosslinked RNA nucleobase. More-over, the absence of ions representing the intact peptidecrosslinked to either adenosine or cytidine supports ourconclusion that U is the actual crosslinked nucleotide.The m/z values of the ions marked with an asteriskconsistently represent the mass of the remaining RNAmoiety added to the mass of the intact crosslinkedpeptide sequence. Thus, the precursor at m/z 642.6 (�3)was unambiguously revealed to be the U1 70K protein,encompassing positions 173–180 (RVLVDVER),

Figure 4. Targeted LC/MS/MS analysis using MRM detection in positive-ion mode. Shown are theMRM traces of m/z 642.6 (�3) to m/z 136.1 (25.0 min) of a crosslink derived from U1 snRNP particlesafter hydrolysis with trypsin and RNase T1 (a) and of m/z 892.3 (�2) to 136.1 (49.5 min) fromcrosslinked [15.5.K-61K-U4atac snRNA] complexes after treatment with chymotrypsin and RNases Aand T1 (b).


crosslinked to a trinucleotide AUC (presumably be-tween positions 29 and 31 in the U1 snRNA). As theresult of incomplete CID fragmentation, we were notable to determine unambiguously the actual crosslinkedamino acid. Nonetheless, CID enabled us (1) to identifythe crosslinked protein as such and (2) to narrow downthe crosslinking site to residues 173–175 (RVL). Whenperforming analyses, especially with the aim of charac-terizing hitherto non-characterized ribonucleoproteinparticles, such information is of importance because itallows us, for example, to identify previously unknownRNA-binding domains. This is of great value evenwithout knowledge of the exact point of crosslinking inthe° peptide.° Figure° 5c° summarizes° the° resultsschematically.Figure° 6a° shows° the° charge-state° deconvoluted

MS/MS spectrum for the putative crosslinked precur-sor at m/z 892.3 (�2) derived from [15.5.K-61K-U4atacsnRNA] particles after hydrolysis with chymotrypsinand RNases A and T1. A b-type fragment-ion seriesfrom b3 to b6 can be observed for the crosslinkedpeptide, revealing the sequence SVLP, as well as severalyp-type ions, those marked with hash marks represent-ing the mass of the remaining peptide moiety added tothe mass of the intact crosslinked RNA sequence (forasterisks see above). The measured precursor masstogether with the losses of adenine (�m � 135) andadenosine (�m � 329) demonstrate that the peptide iscrosslinked through the uracil rather than the adeninebase. The combination of all these results reveals thatthe U4/U6 snRNP specific 61K protein between posi-tions 263 and 273 (SSTSVLPHTGY) is crosslinked to anAU dinucleotide (presumably between positions 43 and44 in the U4atac snRNA). The results are summarizedschematically°in°Figure°6b.These results are consistent with those of our previ-

ous studies in which we sequenced, for the first time,the crosslinked peptide and RNA moieties of theseisolated°crosslinks°by°MALDI-ToF°MS/MS°[22].°Impor-tantly, CID used here for structural investigations hasthe advantage over MALDI-MS/MS that the neutralloss fragmentation (�98; known to be the dominantfragmentation during MALDI-MS/MS experiments forphosphopeptides and which we also observed duringMS/MS of crosslinks) is negligible, so that more intenseion series, particularly those of the crosslinked peptidepart, are obtained.The feasibility study described here has thus proven

that our approach is useful for the detection of un-known crosslinked peptides from a crude mixture and,importantly, for obtaining structural information aboutthe crosslinked peptide and RNA moieties.In general, hydrolysis of the RNA with two endo-

nucleases improves the detection by ESI-MS overpeptide–RNA conjugates with longer RNA moieties,that is, those derived after digestion with only RNaseT1 (see above). The precursor ion scan plot from theU1 snRNP particle treated with trypsin and RNase T1(Figure°3A)°showed°that°non-crosslinked°RNA°oligo-

nucleotides are co-eluting during the entire separa-tion and that defined signals are derived only fromcrosslinks with a non-specific RNase T1 oligonucleo-tide, such as AUC crosslinked to RVLVDVER. More-over, when performing experiments with UV-irradiated[15.5K-61K-U4atac snRNA] complexes treated with en-doproteinase chymotrypsin and ribonuclease RNase T1,we were unable to identify the formerly established 61Kcrosslink between amino acids SSTSVLPHTGY (posi-tions 263–273) and the U4atac T1 oligonucleotideCAUAG°(positions°42–°46)°[22,°43].°Off-line°and°on-lineESI experiments with that particular crosslink purifiedin°a°semi-preparative°manner°[21]°showed°no°signalfrom the crosslink when investigated in the ESI massspectrometer (data not shown). We suggest that thephysicochemical properties of the peptide and RNApart of peptide–RNA heteroconjugates with longerRNA moieties (number of nucleotides 3) are indeedtoo divergent for ESI-MS, at least under our conditions.

Conclusion

We have developed an LC/MS/MS method that allowsthe sensitive and specific analysis of substoichiometricpeptide–RNA oligonucleotide crosslinks in complexmixtures resulting from UV crosslinking experiments ofnative RNP particles and RNP particles reconstituted invitro. By a combination of enzymatic digestion,nanoLC, and highly specific MS experiments we wereable to detect and sequence crosslinks in well-describedtest systems ([15.5K-61K-U4atac snRNA] and U1snRNP°[43,°44]).°The°mass-spectrometric°analysis°on°ahybrid triple quadrupole/linear ion-trap (QqLIT, QTRAP) includes a precursor ion scan for m/z 79 in thenegative-ion mode to selectively detect analytes con-taining a phosphate moiety, followed by a targetedLC/MS/MS analysis using MRM in the positive-ionmode to identify the crosslinked species by their expecteddecomposition to one of the four possible RNA nucleo-bases C=, U=, A=, or G= (m/z 112, 113, 136, 152 in �ve).Our approach combines several layers of specificity:

it removes the bulk of non-crosslinked RNA compo-nents during on-line nanoLC and the configuration ofthe MS analysis excludes the detection of non-crosslinked peptides. In consequence, we detectedhardly any false positives during the analysis. Thetwo-run LC/MS/MS strategy improves the data qualityover a single-run strategy in which a precursor ion scanis combined with a product ion scan as described byWilliamson°et° al.° [39].°This° is°because° it° (1)° admitsdifferent precursor charge states and (2) provides afaster cycle time compared with a full scan, precursorion scan-driven experiment. Thus, MS/MS experimentsare triggered under close-to-optimum conditions,which in turn allows charge-state deconvolution of theresulting product ion spectra.Another benefit is that, as the polarity switch and

product ion scan components are removed from theprecursor ion scan experiment, more cycle time is spent


4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Figure 5. Product ion scan (a) and charge-state deconvoluted MS/MS spectrum (b) form/z 642.6 (�3)obtained during targeted LC/MS/MS analysis of native U1 snRNP particles after hydrolysis withtrypsin and RNase T1. Found peptide and RNA fragment ions are assigned in the spectra. RNAfragmentation°nomenclature°is°according°to°McLuckey°et°al.°[47].°y-type°peptide°fragment-ion°seriesare named yp1 to ypn. Asterisks next to fragments indicate that the measured fragment massesrepresent RNA fragments bound to the intact crosslinked peptide moiety. The insert in (a) shows theenlarged view of the lower m/z region of the spectrum. A schematic representation of the RNA andpeptide sequences of the identified crosslink is given in (c). Observed fragmentation during targetedLC/MS/MS analysis is indicated. For details see text.

Figure 6. Charge-state deconvoluted MS/MS spectrum for m/z 892.3 (�2) obtained during targetedLC/MS/MS analysis of reconstituted [15.5.K-61K-U4atac snRNA] particles after hydrolysis withchymotrypsin and RNases A and T1 (a). The peptide and RNA fragment ions found are assigned inthe°spectrum.°Fragmentation°nomenclature°is°according°to°McLuckey°et°al.°[47].°Hash°marks°next°tofragments indicate that the measured fragment masses represent peptide fragments bound to theintact°crosslinked°RNA°moieties°(for°asterisks°see°legend°to°Figure°5).°A°schematic°representation°ofthe RNA and peptide sequences of the identified crosslink is given in (b). Observed fragmentationduring targeted LC/MS/MS analysis is indicated. For details see text.


on the precursor ion scan itself and on the data-dependent high-resolution linear ion-trap scans. As aconsequence, a more complete “molecular weight ver-sus retention time” map (“heat map”) of the sample isgenerated. The information obtained can be comparedwith corroborating evidence from previous experi-ments or with a list of computationally predictedcrosslinks generated before the final MRM experimentin which MS/MS full scans are collected.In conclusion, the analytical method is highly selec-

tive. Given that the starting material used was about 5%of°that°used°for°previous°experiments°[21,°22],°it°alsopossesses excellent sensitivity. Moreover, detailed andreliable structural information of the crosslinks is ob-tained from the combination of precursor ion scan,multiple reaction monitoring, and collision cell CID-MS/MS experiments.In summary, we conclude that our ESI approach is

highly suitable for the rapid detection of unknowncrosslinked peptides from crude mixtures and the sub-sequent identification of the crosslinked protein byMRM-driven LC/MS/MS acquisition. Note that theentire approach is optimized for detection ofcrosslinked proteins. Dual ribonuclease digestionclearly favors the detection of the crosslinks in crudemixtures. Because of the short length of the RNAmoiety obtained by this approach, identification of thecorresponding crosslinking site on the RNA is morechallenging at this stage.

AcknowledgmentsThe authors thank Reinhard Lührmann, Department of CellularBiochemistry, MPI for Biophysical Chemistry, for providing HeLanuclear extract and continuously supporting this work. MonikaRaabe and Uwe Ple�mann are acknowledged for their excellenthelp in crosslink purification. E. Kühn-Hölsken is funded by thePeter und Traudl Engelhorn-Stiftung. This work is supported by aYouth Investigator Program grant from the EURASNET (withinthe 6th EU framework) to H. Urlaub.

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