B The Author(s), 2018. This article is an open access publication J. Am. Soc. Mass Spectrom. (2018) 29:1700Y1705 DOI: 10.1007/s13361-018-1978-y RESEARCH ARTICLE Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin 1 2 1 1 2,3 Federico Floris, Lionel Chiron, Alice M. Lynch, Mark P. Barrow, Marc-André Delsuc, Peter B. O’Connor Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK CASC4DE, 20 Avenue du Neuhof, 67100, Strasbourg, France Institut de Génétique et de Biologie Moléculaire et Cellulaire, Institut National de la Santé et de la Recherche, U596; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Université de Strasbourg, 67404, Illkirch-Graffenstaden, France Abstract. Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and frag- ment ions by modulating precursor ions system- atically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier trans- form ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmen- tation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Keywords: FT-ICR MS, 2DMS, Top-down proteomics Received: 6 February 2018/Revised: 13 April 2018/Accepted: 21 April 2018/Published Online: 4 June 2018 often leading to a low sequence coverage of the protein under Introduction analysis [4, 5]. The ultra-high resolving power (RP) and mass he complexity of protein forms has expanded the need for accuracy of Fourier transform ion cyclotron resonance (FT- Tproteomics [1, 2], and technologies such as mass spec- ICR) MS [6, 7] alleviate some of the TDP technical limitations, trometry (MS) offer a leading platform for characterization of allowing unambiguous characterization of overlapping isotopic such macromolecules . Studying proteins through MS in distributions and of small mass shifts due to post-translational their entirety (Top-Down, TDP), rather than from smaller pep- modifications (PTM) [4, 8]. tides obtained through their enzymatic digestion (Bottom-Up FT-ICR MS can be used to investigate multiple analytes or Proteomics, BUP), offers the highest amount of structural charge states in a single two-dimensional (2D) MS experiment, information, but constitutes a more challenging experiment generating a 2D mass spectrum that retains all the MS/MS information from all the precursors in the sample simultaneous- ly [9, 10]. 2DMS was developed in the 1980’s[11–14]but was Electronic supplementary material The online version of this article (https:// not pursued after because of computational limitations . doi.org/10.1007/s13361-018-1978-y) contains supplementary material, which With new developments in computer technology and FTMS, is available to authorized users. the technique was updated with a novel processing software, Correspondence to: Peter O’Connor; e-mail: firstname.lastname@example.org SPIKE , cutting-edge algorithms for the de-noising of 2D F. Floris et al.: Application of MS/2DMS to Top-down Deep Sequencing of CaM 1701 mass spectra such as urQRd , and computational and Argon gas. IRMPD was achieved using a continuous wave, 25 tuning optimizations [19, 20]. 2DMS is becoming an efficient W, CO laser (Synrad Inc., Washington, USA) held at 70% of its analytical tool for the analysis of small molecules , macro- power output. IR photons were produced at a wavelength of molecules [22, 23], and proteomics studies [24, 25]. The tech- 10.6 μm and pulsed into the ICR cell for 0.3-0.5 s prior to nique has also been expanded for use in linear ion traps  detection. ECD was performed generating electrons from a and applications in polymer analysis . heated hollow cathode (1.5 A) and pulsating them at 10 V into 2DMS has recently been explored for top-down proteomics the ICR-cell for 0.2 s prior to detection (ECD bias 1.5-2.0 V). in a comparative study with 1D MS/MS, using calmodulin CAD-MS/IRMPD-2DMS and CAD-MS/ECD-2DMS (CaM) as a model, and infrared multiphoton dissociation spectra were acquired respectively with 2048 scans of 512k (IRMPD) as fragmentation technique . The study, later data-points and 1024 scans of 2M data points over a mass range implemented with electron-capture dissociation (ECD) , of m/z 368.2-3000 on the vertical axis and m/z 147.5-3000 on showed comparable results between the one-dimensional and the horizontal axis; total time of acquisition was ~80 min per two-dimensional analyses, with a considerable saving in time- experiment. The 2D mass spectra were processed using SPIKE, and sample-consumption for the latter. Although the study dem- and de-noised with urQRd (k =20). onstrated the suitability of 2DMS for TDP, the reported ~23% See Table S.1 for specific details about the parameters used cleavage coverage of CaM initiated interest to develop a method for the acquisition of the two-dimensional mass spectra and for deep sequencing of proteins so as to achieve higher overall their one-dimensional control spectra. inter-residue bond cleavage coverage, particularly into the inte- rior regions of the protein which often show limited fragmenta- tion. This technique is called tandem two-dimensional mass Results spectrometry (MS/2DMS) . In MS/2DMS a charge state of interest of the protein under analysis is selected through quadru- Figure 2 shows the results of the CAD-MS/IRMPD-2DMS and polar isolation and fragmented in a collision cell (e.g. using CAD-MS/ECD-2DMS analyses of CaM. The resulting 2D collisionally-activated dissociation, CAD). Subsequently, the mass spectra are reported respectively in figures 2.a and 2.e. primary fragments are sent to the ICR-cell for simultaneous The autocorrelation line is extracted from the CAD-MS/ fragmentation with 2DMS, generating a 2D mass spectrum IRMPD-2DMS mass spectrum and reported in Figure 2.b, containing information equivalent to MS of all primary frag- showing a large number of b/y ions, neutral losses, and internal ment ions previously generated in the collision cell. The fragments (reported in percentage): typical ion fragments gen- workflow of MS/2DMS is shown in Figure 1. Deeper investi- erated by techniques such as CAD. gation of macromolecules can be obtained by adding fragmen- Figure 2.c shows the extraction of a horizontal ion scan, tation steps before introduction of the (fragmented) analytes in corresponding to the fragmentation pattern of the CAD frag- n 5+ the ICR-cell; this further technique is called MS /2DMS. ment b (m/z 1206.3786), generated during the fragmenta- In this work, MS/2DMS is applied to calmodulin to achieve tion period inside the ICR-cell. Many b and internal ions and extensive inter-residue fragmentation in a single TDP 2DMS neutral losses are recognized in the spectrum, as expected from 5+ experiment using CAD in the collision cell for the first stage of IRMPD. The horizontal scan of ion b was calibrated using fragmentation and either ECD or IRMPD for the second stage. the theoretical m/z of the recognized fragments, and the obtain- ed fitting parameters were used to calibrate the entire two- dimensional mass spectrum with a quadratic equation. Frag- ment ions could be assigned with a mass accuracy of 0.21±0.98 Experimental ppm. Horizontal fragment ion scans were extracted for all the Materials successfully assigned precursors, and their fragmentation pat- terns assigned and used to calculate the cleavage coverage of Calmodulin from bovine testes was purchased from Sigma- CaM with the CAD-MS/IRMPD-2DMS MS/2DMS experi- Aldrich (Dorset, UK). HPLC grade methanol and formic acid ment, corresponding to ~41%. (HAc), were obtained from Fisher Scientific (Loughborough, A vertical scan is extracted and shown in Figure 2.d. This UK). Water was purified by a Millipore Direct-Q purification mass spectrum shows all the precursors that generate the ion b system (Millipore, Nottingham, UK). (m/z 1070.5000) during the fragmentation period in the ICR- CaM (0.4 μM) was dissolved in a 75:25 water/methanol cell. Figure 2.d shows about 15 precursors, which can only be (v/v) solution with 0.3% (v/v) HAc. 14+ N-terminal precursor ions and the molecular ion MH of CaM. Precursor ions were assigned through cross-correlation Instrumentation with the autocorrelation line. Signals due to experimental noise FT-ICR MS was performed on a 12 T Bruker solariX FT-ICR are labelled with a star symbol (*). The b ions assigned through mass spectrometer (Bruker Daltonik GmbH, Bremen, Germa- the extraction of the vertical ion scan are labelled in Figure 2.b ny), using an electrospray ionization (ESI) source. In-source with a pentagon. dissociation was used to remove salt adducts. CAD was per- A similar analysis has been performed for the spectrum of formed by accelerating the ions to a hexapole collision cell with Figure 2.e, and it is detailed in the Supporting Information (see 1702 F. Floris et al.: Application of MS/2DMS to Top-down Deep Sequencing of CaM Figure 1. In MS/2DMS, a charge state of interest of the protein under analysis is firstly selected using quadrupolar isolation. The isolated ion species is then fragmented by acceleration into the collision cell using CAD), and the generated CAD-fragments are sent into the ICR-cell for two-dimensional mass spectrometry analysis using IRMPD or ECD as fragmentation techniques. A two- dimensional mass spectrum is generated, containing all the IRMPD/ECD fragmentation patterns of the CAD-fragments, constituting information equivalent to MS experiments of each and every primary CAD-fragment ion also Table S.3 and Figure S.3). The cleavage coverage for the fragmentation efficiency of the primary fragmentation should be CAD-MS/ECD-2DMS experiment is ~33% high. A dissociation technique with high fragmentation efficien- Combination of the data obtained with the CAD-MS/ cy will generate abundant primary fragment ions, which can be IRMPD-2DMS and CAD-MS/ECD-2DMS MS/2DMS exper- further fragmented in the ICR-cell. iments of CaM led to a cumulative cleavage coverage of ~42%. Investigation of the horizontal scans represents the equivalent 3 2 of adding MS information to the MS information provided by the analysis of the autocorrelation line. The cleavage coverage map of Figure 3.a shows in red the MS information obtained Discussion with IRMPD as secondary fragmentation technique. Many cleavages occur in the same sites as the primary CAD fragmen- The tandem two-dimensional mass spectrometric analysis of tation (due to the similarity of the dissociation techniques). CaM generated two 2D mass spectra retaining information However, further fragmentation with IRMPD increased the equivalent to MS using CAD as primary fragmentation and cleavage coverage of the protein by ~9%. On the other hand, IRMPD or ECD as secondary fragmentation techniques. ECD did not produce extensive information. It is hypothesized Interpretation of these spectra relies on the analysis of the that such result is due to the low fragmentation efficiency of autocorrelation line, bearing information equivalent to CAD ECDcomparedtoIRMPD.Standard1DECD FT-ICR MS/MS MS/MS, and the extraction of a horizontal line for each primary experiments require a high number of transient accumulations fragment, showing their fragmentation patterns and constituting (with an increase of signal-to-noise ratio, S/N, proportional to the information equivalent to MS . A cleavage coverage map has square root of the number of scans) in order to visualize frag- been built for each analysis, reporting in blue the fragments ments over the limits of detection. In 2DMS, transients cannot be assigned to the autocorrelation line, and in orange (IRMPD) accumulated to improve S/N in the horizontal dimension, but and green (ECD) the fragments assigned to the horizontal ion every iteration of the pulse programme used to obtain the scans (Figure 3). Similar to what is reported for the TDP 2D precursor/fragment correlation (scans in t ) increases the vertical IRMPD MS studies of CaM, the cleavage sites are prevalent in resolution. Finally, ECD generally requires a high precursor ion proximity of the protein termini. Such phenomenon is due to the abundance to produce high-quality tandem mass spectra. Opti- similarity of IRMPD and CAD, used here as primary fragmen- mization of the fragmentation efficiency of the primary dissoci- tation, as ergodic processes. In MS/2DMS the primary fragments ation and/or better storage of the primary fragments in the ICR- are further fragmented using IRMPD or ECD, allowing more cell are relevant parameters on this purpose. Analysis of the detailed characterization of the protein. However, if no primary horizontal ion scans extracted from the CAD-MS/ECD-2DMS fragments are generated from the most internal parts of the spectrum increased the cleavage coverage of the protein by ~1% protein, further fragmentation will not cover missing cleavages. (Figure 5.b). Further studies are in progress to increase the A method to improve the cleavage coverage on this purpose efficiency of ECD in TDP 2DMS. would involve protein unfolding (e.g. through supercharging), or Further information about the protein structure (and disso- the use of a “non-ergodic” fragmentation such as electron- ciation mechanisms) can be obtained by extracting the vertical transfer dissociation before the ICR-cell. It is important to notice (precursor) ion scans. Extracting a vertical ion scan from a 2D that, in order to obtain extensive secondary fragmentations, the F. Floris et al.: Application of MS/2DMS to Top-down Deep Sequencing of CaM 1703 Figure 2. CAD-MS/ECD-2DMS and CAD-MS/IRMPD-2DMS of calmodulin in denaturing conditions. (a) Two-dimensional mass 5+ spectrum for the CAD-MS/IRMPD-2DMS analysis of CaM. (b) Autocorrelation line. (c) Fragment ion scan of the CAD-ion b .(d) Vertical ion scan for the ion b . The assigned ions are labelled on the autocorrelation line (spectrum b) with a pentagon. (e)2D mass 5+ spectrum from the analysis of CaM with CAD-MS/ECD-2DMS. (f) Fragment ion scan of the ion b mass spectrum produces a spectrum of all the precursors that Figure 2.d shows an example with the secondary IRMPD generated the fragment of interest. In MS/2DMS precursor ions ion b , whose precursors can only be other b ions (or even- are primary fragments, i.e. fragments that retain a protein tually any higher a ion, generated by a secondary fragmen- terminus (C- or N-), or internal fragments. Fragment ions that tation path of CAD) and the remaining unfragmented mo- retain a terminus (mainly b/y in case of IRMPD, or c/z for lecular ion. Vertical ion scans are a useful feature of 2DMS, ECD) can be generated only by a precursor with the same but their resolution is often much lower compared to the terminus. This is particularly important for de novo sequenc- horizontal dimension because of their proportional depen- ing with MS/2DMS, for which, once a secondary fragment dence to the number of experimental scans, hence to the ion has been identified, extraction of its vertical ion scan will experimental time . Recent developments in data acqui- show precursors that can have the same terminus. In MS/ sition for 2DMS allow improvement of the vertical resolu- 2DMS precursor ion scans have the potential to easily dis- tion without impairing acquisition times, although increas- criminate primary fragments based on their termini. ing processing times . 1704 F. Floris et al.: Application of MS/2DMS to Top-down Deep Sequencing of CaM Finally, although MS/2DMS still has room for improve- ment, its use in the TDP analysis of CaM raised the cleavage coverage compared to 2D IRMPD MS. This study represents a further step in the analysis of CaM by two-dimensional mass spectrometry. Including previously published BUP and TDP 2DMS data [28, 29], and the MS/2DMS data herein, the aggregate, cumulative cleavage coverage of calmodulin is now ~76%. Conclusions MS/2DMS is a promising tool for deep sequencing of proteins, providing a new fragmentation tool for top-down proteomics. In this work, the cleavage coverage of calmodulin increased from ~ 23% to ~ 42% compared to the top-down 2DMS of the protein alone. Acknowledgements The authors want to thank EPSRC (grants J003022/1 and N021630/1), BBSRC (P021875/1), and Bruker Daltonics, UK, for funding, and Andrew Soulby, Maria van Agthoven, Figure 3. Cleavage coverage maps for the CAD-MS/IRMPD- Hayley Simon, Alice Lynch, and Chris Wootton for helpful 2DMS and CAD-MS/ECD-2DMS analysis of CaM in denaturing conversations. conditions. Vertical lines indicate cleavages from internal frag- ments. The total cleavage coverage for the MS/2DMS analysis Open Access of CaM is ~42% This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// Notably, MS/2DMS experiments have a longer acquisition creativecommons.org/licenses/by/4.0/), which permits unre- time compared to standard 2DMS: ~80 min compared to ~20 stricted use, distribution, and reproduction in any medium, min, because in TDP, primary fragment ions will overlap provided you give appropriate credit to the original author(s) heavily in m/z. Thus, the required vertical resolution, which and the source, provide a link to the Creative Commons is higher for MS/2DMS compared to TDP 2DMS. license, and indicate if changes were made. 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