To Super-Resolution Mass Spectrometry

168 CHIMIA 2014, 68, Nr. 3 Mass spectroMetry in switzerland

doi:10.2533/chimia.2014.168 Chimia 68 (2014) 168–174 © Schweizerische Chemische Gesellschaft From High- to Super-resolution Mass Spectrometry

Yury O. Tsybin*

Abstract: High-resolution mass spectrometry (MS) is indispensable for the molecular-level analysis of biological and environmental samples with great intra- and inter-molecular complexity. Here, we summarize developments in Fourier transform mass spectrometry (FTMS), the flagship of high-resolution MS techniques, accomplished in our laboratory. Particularly, we describe the recent and envisioned progress in structural analysis of: i) isolated large proteins and their simple mixtures, with a focus on monoclonal antibodies, via top–down, middle–down, and extended bottom–up mass spectrometry; ii) complex protein mixtures and proteomes via extended bottom–up proteomics; and iii) crude oil fractions and similar complex molecular mixtures. Despite the unequivocal success in molecular structural analysis, the demonstrated results clearly indicate that the compromise between MS acquisition speed (throughput) and achievable resolution level inhibits further advances of MS applications in the areas related to life, environmental, and material sciences. To further advance beyond state-of-the-art FTMS capabilities in these areas, we present the technique of super-resolution mass spectrometry that has been pioneered by our laboratory. Keywords: Extended bottom–up proteomics · Filter diagonalization method (FDM) · Fourier transform mass spectrometry (FTMS) · Middle–down proteomics · Petroleomics · Resolution · Super-resolution mass spectrometry · Top–down proteomics

Introduction based metabolomics and proteomics.[2] tion (200–400 ms ion detection time) and Nevertheless, the gap between the state-of- above. Significantly higher demands are Mass spectrometry (MS) is the most the-art MS performance and the analytical imposed on quantitative peptide and pro- sensitive and specific analytical tech- demands imposed by the complexity of tein analysis (e.g. 500’000 resolution and nique for molecular and macromolecular molecular systems of interest has not yet the corresponding 1.5 s ion detection time structural analysis.[1] The rapidly improv- been bridged.[3,4] One of the limiting fac- in mass defect-based isobaric tags meth- ing MS analytical characteristics aim to tors that delays further advancement in ods), as well as complex molecular mix- match the growing demands of molecular MS-based applications is the throughput ture analysis (e.g. petroleomics, vide infra) analysis in life, material, and environmen- of analysis in experiments with time con- and peptide isotopic fine structure analysis tal sciences. The current level of MS per- straints. Particularly, high-resolution MS (demands exceed 1’000’000 resolution and formance enables speciation of extremely data should be acquired faster and without 3 s ion detection time). As a result, other complex mixtures of organic and inorganic sacrificing spectral dynamic range. Indeed, MS analytical characteristics, e.g. mass ac- molecules needed for environmental, e.g. sample complexity in modern MS applica- curacy, dynamic range and sensitivity, suf- in MS-based petroleomics and dissolved tions requires a certain level of resolution fer from the compromise between speed organic matter analysis, as well as mate- defined by the ability to distinguish iso- and resolution. Furthermore, although rial sciences applications. For life sciences baric compounds, e.g. species with simi- very high, the ultimate levels of resolution applications, high-performance MS is in- lar mass-to-charge (m/z) ratios. From the achieved in the MS experiments without dispensable for identification and in-depth practical consideration, resolution is typi- time constraints (e.g. petroleomics) remain characterization, including quantitation cally defined as the ratio of the peak loca- limited, with a few exceptions.[5] Therefore, and modifications mapping, of metabolites tion on the m/z scale to the peak width at increasing the overall resolution level may and proteins embedded in complex molec- the half of the peak maximum (FWHM). open new horizons in molecular structure ular environments, e.g. as realized in MS- The narrower the peak – the higher the analysis, as well as provide insights into resolution. Resolution above 30’000 may the related fundamental molecular physics be referred to as high resolution, albeit res- and chemistry.[6] olution classification depends on the type Herein, we describe an ongoing ef- of a mass spectrometer for which it is de- fort undertaken at the Biomolecular Mass fined. Although for most applications the Spectrometry Laboratory (LSMB) at required levels of resolution can now be Ecole Polytechnique Fédérale de Lausanne achieved with high-resolution MS instru- (EPFL) to accelerate state-of-the-art high- ments, the throughput of these measure- resolution FTMS and to develop super- ments is not yet sufficient. For example, resolution mass spectrometry (SRMS) that when Fourier transform mass spectrometry addresses the above described limitations *Correspondence: Prof. Dr. Y. O. Tsybin Biomolecular Mass Spectrometry Laboratory (FTMS, vide infra) is employed, qualita- to advance MS-based applications in life, Ecole Polytechnique Fédérale de Lausanne tive peptide analysis typically requires material, and environmental sciences. To EPFL BCH 4307 resolution of 15–30’000 (50–100 ms ion achieve these advances, innovations in the CH-1015 Lausanne detection time), whereas protein struc- allied fields of signal processing, funda- Tel.: + 41 21 693 97 51 E-mail: [email protected] tural analysis asks for 60–120’000 resolu- mental ion physics, and analytical instru- Mass spectroMetry in switzerland CHIMIA 2014, 68, Nr. 3 169

mentation are required. Finally, following dal. Deviations from sinusoidal nature of FTMS application areas that demonstrate the motto “to break the rules you must first transient components lead to formation the need for SRMS development. master them” of a famous Swiss watch- of additional features in the Fourier spec- maker we report our achievements in the tra, e.g. high-order frequency harmonics corresponding fields of targeted molecular and sidebands around the peaks of inter- Advancing FTMS-based analysis, proteomics, and petroleomics est, which may be manifested as artifacts Applications for Molecular obtained with the state-of-the-art FTMS in mass spectra.[16] The presence of these Structure Analysis technology. components can be minimized by the ratio- nal design of the mass analyzers that aim Analytical characteristics of FTMS for generation and analysis of the principal allow for its application not only for the Fourier Transform Mass harmonic or principal frequency multiple. analysis of molecular mixtures separated Spectrometry: The Starting Point Typically, electrodes for induced current in solution or gas phase prior to injection detection in FTMS are made sufficiently into the mass analyzer, but also for the di- Fourier transform mass spectrometry large to integrate ion motion over a given rect analysis of extremely complex molec- (FTMS) provides superior analytical char- path. For instance, in FT-ICR MS, most ular mixtures. However, the performance acteristics, specifically resolution and mass ICR cells feature a pair of 900 electrodes of both these approaches requires further accuracy, to all other MS techniques.[7–10] for ion detection and work most efficiently improvement. Below we summarize our Additionally, FTMS uniquely offers an at the first harmonic or first frequency mul- recent advances in methods and techniques intriguing capability for further improve- tiple ion detection. enabling progress of these FTMS applica- ment through advances in the generation On the other hand, the application of tions. and treatment of time-domain ion signals, FT signal processing to the sinusoidal sig- commonly referred to as transients. The nals in modern FTMS results in a limita- Proteome Analysis: Extended two widely employed FTMS instruments tion on the resolution level achieved in a Bottom–Up Proteomics are those based on an ion cyclotron reso- given time period of ion signal detection The standard method of complex bio- nance (ICR) and an orbitrap ion trap (or (FT uncertainty principle).[17] The FT reso- logical mixtures, e.g. proteomes, analysis cells). The first operates in a homogenous lution limitation in modern FTMS is at the by MS is through bottom–up proteomics magnetic field environment, whereas the level of the achieved resolution equal to (Fig. 1). The power of this approach lies second is an electrostatic field-based mass about one unit per 2–4 periods of ion os- in the excellent analytical characteristics analyzer.[11] The third, emerging type of cillation. Therefore, to achieve a resolution of MS and liquid chromatography (LC) FTMS mass analyzer is an electrostatic ion of 100’000 the recorded transient should for analysis of isolated short (10–30 ami- trap employing a multi-reflection time-of- contain a substantial number (200’000– no acids in length) peptides produced via flight (TOF) mass analyzer.[12–14] The main 400’000) of ion oscillation periods. The well-established enzymatic digestion pro- competitors of FTMS instruments are the provided estimation of resolution per pe- tocols. However, the extremely high num- state-of-the-art reflectron TOF MS with riod is based on the transient of a sufficient ber of components and more than 10 orders single and multiple turns of ions in their length, required for determination of ion of magnitude dynamic range of protein flight path between an ion source and the oscillation frequency. A key question is: concentrations in the real-life biological ion collision-based detector. They provide How should the FT resolution limitation samples significantly reduce the currently up to 50’000 resolution at 1000 m/z with achieved with modern FTMS instruments achieved depth of the MS analysis. Here, 10–100 Hz scan rate. Note, spectral dy- best be overcome to accelerate the acqui- increased throughput of the high-resolu- namic range is a function of the scan rate in sition speed of high-resolution MS data? tion MS analysis potentially offered by TOF MS and thus accumulation of a high The approach advocated by our laboratory SRMS would be of great interest. quality mass spectrum (dynamic range is comprised of both MS hardware (mass On the other hand, already provided by above 3 orders of magnitude) is time-con- analyzers) and software (signal processing) commercial instruments, LC-based sepa- suming. Recent implementation of high- innovations, the synergy of which should ration and high-resolution MS capabilities resolution (up to 10 bit on the vertical, am- result in development of super-resolution enable efficient and rapid mass analysis of plitude, scale) and high-frequency digitiz- mass spectrometry (SRMS), vide infra. longer (30–70 amino acids) peptides. We ers has significantly increased the achiev- Before the SRMS description we will pres- have recently suggested referring to the able dynamic range of TOF MS. Some of ent some of the currently most promising proteome analysis based on identification the modern ion detectors employ the power of charge sensitive devices for ion detec- Fig. 1. An overview of tion in TOF MS, which demonstrates im- mass spectrometry- proved performance specifically for heavy based proteomics ion detection, particularly important for strategies. intact protein level mass analysis.[15] The underlying common principle of mass analysis in FTMS instruments is Top-Down MS the generation and detection of periodic Middle-Down MS >15 kDa Extended ion motion. The measured transients can Bottom-Up MS 7-15 kDa be Fourier transformed to yield the corre- Bottom-Up MS 3-7 kDa sponding frequency spectra. Application of the known relations between the fre- < 3 kDa kDa quencies of ion oscillations and their m/z values enables generation of the mass Throughput, digestion artifacts spectra. The described standard procedure of FTMS mass spectra generation Precursorion size, charge state works most efficiently when ion signal components in transients are sinusoi- Proteoform ID 170 CHIMIA 2014, 68, Nr. 3 Mass spectroMetry in switzerland

of these long peptides as extended bottom– and rapid (1 h digestion reaction) produc- and protein complex stoichiometry. For up proteomics (Fig. 1).[18,19] Increasing the tion of 3–7 kDa peptides. A particular example, MS/MS of intact IgGs and their size of enzymatically derived peptides advantage of Sap9 compared with other large subunits is of a particular importance effectively reduces the total number of enzymes is its comparable-level activity for their quality control and for improving peptides produced by each protein, which in a wide range of pH values. It is known the drug discovery process. With these in- may significantly reduce the complexity that enzymatic digestion under pH condi- centives in mind, we extended the range of peptide mixtures submitted for LC-MS tions typical for trypsin, e.g. pH 7–8, may of MS/MS applications to 150 kDa intact analysis and increase confidence in protein induce protein artifacts, such as deamida- monoclonal antibodies structural analysis, identification with longer peptides (higher tion, whereas performing digestion at pH first on a high-resolution time-of-flight protein sequence coverage provided by ~5 reduces the presence of such artifacts. (TOF) MS and then on a standard and a single peptide). Application of SRMS These characteristics of Sap9 performance high-field Orbitrap FTMS (Fig. 2).[21,22] methodology may further increase the are particularly beneficial for the targeted By applying electron transfer dissociation throughput of MS analysis, which neces- protein structural analysis, e.g. of mono- (ETD) for MS/MS of intact protein ions, sarily should be a high-resolution one, to clonal antibodies, immunoglobulins G we not only achieved protein sequence accommodate the needs for long peptide (IgGs). On the other hand, the non-specific coverage exceeding previous (limited) at- 13C-isotopic-level analysis. The remaining nature of Sap9 reduces the efficiency of its tempts of top-down collision induced dis- bottleneck is how best to produce these application to large-scale proteomics stud- sociation (CID) MS/MS-based analysis of long peptides. ies. Therefore, further optimization of the monoclonal antibodies, but also increased It is known that restricted by digestion experimental parameters is required. the molecular weight of proteins frag- time proteolysis with typically employed Further increasing size, above 7 kDa, of mented with ETD MS/MS in general.[27] enzymes, such as trypsin, produces long proteolytically-derived peptides requires These results have further contributed to peptides. However, the reproducibility and significant modification of LC, MS, and the already significant interest in the MS- substrate specificity of these reactions have MS/MS parameters to deliver optimum based structural analysis of monoclonal not yet been sufficiently studied, whereas performance. MS-based approach that tar- antibodies. In follow-up work, Marshall preliminary studies demonstrate funda- gets analysis of such peptides, or more cor- and co-workers achieved similar sequence mental limitations of this approach. Thus, rectly said – proteins (polypeptides above coverage of monoclonal antibodies with we first attempted to answer the following 5 kDa are typically referred to as proteins), electron capture dissociation (ECD) on questions: what should be the specificity is termed middle–down mass spectrometry an FT-ICR MS and demonstrated isoto- of a protease for optimum production of (proteomics) (Fig. 1). From the analytical pic-level resolution of antibodies.[28,29] 3–7 kDa peptides? And what proteases of (MS) perspective, the middle–down ap- Brodbelt and co-workers matched these enzymatic or chemical nature exist that proach overlaps with the top–down one, as results by advancing high-resolution would match the determined optimum described below. Orbitrap FTMS analysis of antibodies by specificity rules? To address the first ques- achieving their isotopic-level resolution[30] tion we performed an extensive bioinfor- Protein Analysis: Top–Down and and attempting to fragment their intact ions matics investigation, which demonstrated Middle–Down Proteomics with a promising method of 193 nm ultra- that a specific cleavage at a given amino Top–down MS or proteomics refers violet photo-dissociation (UVPD).[31] acid or a doublet of amino acids will not to tandem mass spectrometry (MS/MS)- Despite a certain level of success produce a sufficient number of 3–7 kDa based analysis of intact proteins or protein achieved with top-down MS of monoclo- unique peptides to represent the complete complexes, as well as their large, typical- nal antibodies, the obtained sequence cov- proteome.[18] On the other hand, combina- ly >15 kDa, subunits (Fig. 1). The com- erage remained at the 33% limit and small tion of cleavages at rare amino acids, e.g. plexity of MS and MS/MS data obtained size modifications, e.g. oxidation, could Trp or Met, or doublets of amino acids, during analysis of such large molecules not be efficiently identified with this ap- e.g. dibasic sites, with a limited tendency dictates the need for high-resolution and proach. To address these issues, we reduced toward non-specific cleavages may indeed high mass accuracy MS performance.[21,22] the size of antibodies by their site-specific provide the required pools of peptides. Theoretically, the top–down approach is and artifact-free digestion in a hinge region In our laboratory, we implemented and the ultimate goal in MS-based protein and with a dedicated enzyme, IdeS (Fig. 2).[25] characterized two workflows for produc- proteome analysis – as it allows for proteo- Depending on the extent of the additional tion of long peptides: i) a workflow based form-level molecular analysis.[23] Already reduction of disulfide bonds the resulting on digestion with in-house expressed and at the MS level information on a protein antibody subunits could be of 100 kDa, purified enzyme, secreted aspartic prote- sample may be employed to quantitatively 50 kDa, or 25 kDa. Application of ETD ase Sap9;[19] and ii) a workflow based on describe proteoform heterogeneity, but and higher collisional energy (HCD) MS/ proteolytic reactions with chemicals, e.g. only when the mass difference between MS to these large fragments on high-field CNBr.[20] Interestingly, despite a large the proteoforms is sufficiently large. In Orbitrap FTMS resulted in increased se- number of studies described in the prior our work, we employed this MS-level ap- quence coverage (up to 70%) and improved literature on protein digestion with chemi- proach for monitoring glycosylation pro- modifications analysis, including the un- cal reagents, there have been no reported files following stable and transient mono- ambiguous assignment of oxidation sites. attempts of their application for protein clonal antibody production and storage.[24] Importantly, analysis of 25–50 kDa instead mixture analysis. Both workflows demon- Avoiding the enzymatic or chemical di- of 150 kDa proteins simplifies the related strated intriguing capabilities for extend- gestion step minimizes the introduction LC-MS/MS experiment and substantially ing bottom–up proteomics to the analysis of possible artifacts during sample prepa- increases the performance for the analysis of longer peptides. ration, e.g. deamidation, and reveals the of antibody mixtures. Due to the involve- The specificity of Sap9 enzyme to connectivity of protein complex subgroups ment of an enzymatic digestion step to the dibasic sites, as originally suggested and modifications.[25,26] Adding a MS/MS the procedure, the described analysis can in the literature, has been only partially step to structural analysis of proteins and be referred to as middle–down approach, confirmed by our large-scale studies.[19] protein complexes potentially provides whereas the size of proteins, 25–100 kDa, However, combining dibasic specificity protein primary structure information justifies classification of this approach as with unspecific cleavages enabled efficient (sequence and location of modifications) top–down. Mass spectroMetry in switzerland CHIMIA 2014, 68, Nr. 3 171

FT-ICR MS-based petroleomics, such as MS data mass spectra recalibration procedures, to 150 kDa Orbitrap FTMS is not possible due to the differences in fundamentals of ion motion Intact IgG between these two FTMS technologies. Therefore, the main steps in the LSMB IdeS FT-ICR MS workflow include custom-developed al- 30 min digestion gorithms that consider the particular characteristics of Orbitrap FTMS data: i) gen- 2x50 kDa Data eration and acquisition of transients with F(ab’)2 LC-MS/MS analysis extended lengths, up to 3–6 s. Transient data treatment in absorption mode FT 50 kDa ETD/ECD Standard spectral representation is particularly use- Fc CID/HCD & high-field ful, thus the use of the eFT algorithm is en- Chemical abc Orbitrap FTMS [40] denaturation, couraged; ii) thresholding of raw mass reduction spectral data by distinguishing analyte and noise distributions via plotting peak Fd MS/MS data 3x25 kDa High-resolution component density against the decimal reflectron logarithm of peak intensity;[41] iii) iterative Fc/2 TOF MS recalibration of mass spectral data with an xyz empirical estimation of the mass calibra- Lc tion function, which takes into account not only the m/z values of the peaks but also Fig. 2. High-resolution mass spectrometry: top–down and middle–down mass spectrometry work- their abundances;[40] iv) the obtained mass flows. accuracy values allow unambiguous peak assignment and v) data visualization fol- In addition to analysis of monoclonal method that is particularly useful once the lowing the hexagonal class representation antibodies, in different collaborations we reference information from direct infusion method based on relative abundance versus have shown the utility of the top–down high-resolution MS data is already avail- compound classes plot.[16] The described MS approach in the analysis of protein– able for a given sample type. workflow has been successfully applied to drug complexes and clinical studies. In Until recently, only high-resolution high-field Orbitrap FTMS-based analysis collaboration with Dyson and co-workers, FT-ICR MS was capable of delivering of crude oil fractions of low to medium we investigated the binding of anti-cancer the level of resolution and mass accuracy complexity.[39] Notably, FTMS transients organometallic drugs and drug candidates required for petroleomics. At LSMB we of only 1.5 s duration were employed in to small proteins, which revealed the in- have developed an alternative workflow, this pioneering study. Increased sample sights of the mode of action of these small based on a high-field Orbitrap FTMS, that complexity requires measurement of high- molecule drugs.[32–34] In collaboration with delivers petroleomics-grade performance er resolution FTMS data, which is now Hochstrasser and co-workers, we applied for many related samples without the need readily offered by the acquisition of longer, top–down MS to increase the robustness for an expensive super-conducting magnet up to 3–6 s, transients.[42] Nevertheless, and speed of the amino-acid level modifi- (Fig. 3).[39] Interestingly, direct application further improvements in resolution perfor- cations analysis of clinically relevant pro- of data analysis strategies developed for mance are required to increase the through- teins, starting with hemoglobin and related hemaglobinopathies diagnostics.[35] BOSCAN_Obri_480Ke_500scans_glass #1 RT: 43.37 AV: 1 NL: 1.64E4 T: FTMS + p ESI Full ms [100.00-2000.00] 100 90 MS data Complex Mixture Analysis: 80 APPI/ESI 70 e 60 eFTof 3 s transients

Petroleomics undanc 50 Ab

MS e iv

at 40 Petroleomics generally refers to di- Rel 30 rect infusion high-resolution mass spec- 20 10

0 200 300 400 500 600 700 800 900 1000 trometry of extremely complex molecular m/z [36,37] Zhurov et al., Energy&Fuels, 2013, 2974 mixtures. Most often MS-based petro- Sample High-field leomics deals with crude oil and its frac- Orbitrap FTMS tions. The allied application areas cover Noise filtering environmental sciences (including analy- Sample ID Key characteristics: sis of dissolved organic matter from water Resolution: ~ 900k @ 400 m/z reservoirs) and material sciences (includ- From light to heavy crude oil analysis, Dynamic Range: 3 –5orders ing reaction products of high complexity, high sulfur content RMS Mass Accuracy: ~ 350 ppb e.g. those obtained upon high-pressure Zhurov et al., Anal. Chem. 2014 decomposition and hydrogenation of fullerenes,[38] as well as biofuels analy- Sample Characterization Peak ID Mass accuracy improvement sis). Efficient separation of these complex molecular mixtures by liquid or gas chromatography is often not feasible, making high-resolution MS analysis indispensable. Recent advances in gas-phase ion separation technique, ion mobility (IM) time-of-flight (TOF) MS, led to substantial Zhurov et al., Anal. Chem., 2013, 5311 Kozhinov et al. Anal. Chem., 2013, 6437 progress of this complementary analytical Fig. 3. High-resolution mass spectrometry: Orbitrap FTMS-based petroleomics workflow. 172 CHIMIA 2014, 68, Nr. 3 Mass spectroMetry in switzerland

put of petroleomics experiments and for Fig. 4. Super- analysis of heavy (above 1000 Da) isobaric Super-ResolutionMass Spectrometry resolution mass ions demonstrating less than 1 mDa mass spectrometry: a syn- difference. These advances would be wel- Innovations in MS instrumentation ergy of innovations in come not only for Orbitrap but also for ICR MS instrumentation, 4f ICR FTMS advanced signal FTMS-based petroleomics. processing, and data acquisition. Super-resolution Mass Spectrometry: Pushing the Limits of FTMS Applications

Above, we have presented results in petroleomics as well as top–down, mid- Advanced signal processing dle–down, and extended bottom–up proteomics demonstrating the substantial advances achieved in molecular analysis with modern high-resolution MS. Nevertheless, extremely high sample complexity often exceeds performance of FTMS-based platforms employed in these approaches. Super-resolution mass spectrometry (SRMS) is aimed to advance FTMS by delivering high-resolution MS data faster (at High-performance data acquisition system least 10 times). By our definition, SRMS is an analytical technique that employs super- resolution methods of signal processing for transient time-to-frequency conversion.[43] Therefore, SRMS may surpass FTMS in terms of resolution as the quantitative form of time-to-frequency uncertainty principle of super-resolution methods of signal processing is less strict compared to that of the FT-based signal processing. The current roadmap for SRMS development at out changing the magnetic or electric field acquisition system (National Instruments LSMB (Fig. 4) is based on the synergy strength. For example, we demonstrated electronics based), enabling a high (up of: i) innovations in MS instrumentation, that doubling the frequency (and, thus, the to 100 MHz) frequency sampling rate at with a focus on generation of transients resolution) on a commercial FT-ICR MS substantial vertical resolution (16 bit) and composed of user-defined functions, e.g. is feasible without significant hardware rapid data transfer for acquisition of tran- harmonic frequency multiples; and ii) ad- modifications.[44] Direct detection of high- sients of any length (Fig. 4, bottom panel). vances in signal processing, with a focus on er-order harmonics or frequency multiples The matching signal processing should super-resolution signal processing method has been discussed in the literature,[45] but be developed to maximize the advantages implementation;[43] supported by iii) high- a dedicated implementation on a modern provided by this ICR cell. Our preliminary performance data acquisition systems de- FTMS instrument suitable for biomolecu- results in the corresponding signal pro- velopment for maximizing the information lar analysis has been missing. Recently, cessing development are briefly summa- output and matching the requirements of we implemented the 16-electrode ICR cell rized below. signal processing algorithms. On the other on the proteomics-grade 10 T FT-ICR MS hand, the MS datasets generated follow- (LTQ FTMS) platform that enables the Super-resolution Mass ing the presented workflows (Figs 2 and efficient recording of the quadruple fre- Spectrometry Signal Processing 3), especially petroleomics mass spectra, quency multiple (Fig. 4, top panel).[46] This At the software level, SRMS develop- being extremely complex in m/z and abun- implementation accelerates acquisition of ment involves implementation of non-FT dance directions, are particularly useful for high-resolution FTMS data by four times, and advanced FT methods of signal pro- SRMS development and evaluation. In the providing the expected benefits for molec- cessing for transient analysis and goes following we briefly summarize our main ular and macromolecular structure analy- in two ways: i) signal processing of har- achievements in SRMS development. sis. Can we go beyond this achievement to monic transients with methods that al- further increase the recorded frequency? low the magnitude-mode FT resolution Instrumentation for Advanced To address the objectives discussed limitation to be overcome; and ii) signal FTMS and Super-resolution Mass above, we invented and implemented a processing of transients composed of user- Spectrometry dedicated ICR cell with modified trapping, defined functions. To advance the former A standard approach of accelerating excitation, and space-charge fields.[47] way, we implemented two super-resolution FTMS is to increase the frequency of har- The preliminary data demonstrates the methods of signal processing, the well- monic components in the FTMS transients. envisioned capabilities of this ICR cell developed parameter estimators, filter di- That is typically achieved by increasing for faster high-resolution MS, as well as agonalization method (FDM)[43,48] and a the magnetic field strength in FT-ICR MS for improved analytical characteristics of least squares fitting (LSF)[49] to analyze or electrostatic field strength in Orbitrap standard-speed high-resolution FTMS. the experimental ICR and Orbitrap FTMS FTMS. However, there are opportunities The experimental data were obtained with transients. Fundamentally, the super-reso- for increasing the detected frequency with- the in-house-built high-performance data lution methods, including FDM and LSF, Mass spectroMetry in switzerland CHIMIA 2014, 68, Nr. 3 173

should allow peaks in mass (frequency) mentals development and implementation Scientific for enabling additional features to spectra to be resolved using shorter tran- as an original and complementary analyti- our instruments and overall technical support. sients than required by FT. Indeed, our cal technique to FTMS. Importantly, pre- We are grateful to Bruker Daltonics for sup- data (including the success in implementa- liminary results validate the feasibility of porting our top-down mass spectrometry de- tion of ICR cells providing high frequency this approach, whereas the FTMS applica- velopment initiatives with TOF MS. The work was supported by the Swiss National Science multiples and harmonics) demonstrates tions, including those demonstrated here, Foundation (Projects 200021-125147, 200021- that due to a discrepancy between the FT confirm the need for faster high-resolution 147006, and 128357), the European Research requirements (FT uncertainty principle) on MS. Further development of advanced sig- Council (ERC Starting grant 280271), and the length of the transient signal and the nal processing and high-performance data EPFL. instrumentally achieved coherence of ion acquisition techniques coupled with MS Received: February 13, 2014 motion in the phase space, there is room instrumentation shall lead to a robust and for possible resolution improvement. Our routine SRMS technology. On the other [1] E. Sabido, N. Selevsek, R. Aebersold, Curr. results with FDM and LSF application to hand, exceptional computational power re- Opin. Biotechnol. 2012, 23, 591. experimental ICR and Orbitrap FTMS data quirements for transient data analysis limit [2] Y. O. Tsybin, L. Fornelli, A. N. Kozhinov, A. demonstrate that the gain in resolution per- more rapid development and acceptance of Vorobyev, S. M. Miladinovic, Chimia 2011, 65, 641. formance is governed by spectral irregular- the SRMS technology. The allied FTMS/ [3] R. A. Zubarev, Proteomics 2013, 13, 723. ity. We reported experimental data where SRMS instrumentation development pro- [4] A. Michalski, J. Cox, M. Mann, J. Proteome the gain ranges from a minimum (2-fold) gram at our laboratory led to implementa- Res. 2011, 10, 1785. (Fig. 4, middle panel), to significant (up to tion of the quadruple frequency multiple [5] I. A. Boldin, E. N. Nikolaev, Rapid Commun. 30-fold) values, e.g. as in the analysis of ICR cell and innovation of a novel type of Mass Spectrom. 2011, 25, 122. [6] A. G. Marshall, C. L. Hendrickson, S. D. H. [48] isotopic fine structures of peptides, and ICR cells that demonstrate superior ana- Shi, Anal. Chem. 2002, 74, 252A. in protein quantitation with isotopically- lytical performance to the currently em- [7] A. G. Marshall, C. L. Hendrickson, Ann. Rev. coded isobaric tags with mass defects. To ployed ICR mass analyzers. Anal. Chem. 2008, 1, 579. advance the latter way, the efficient sig- In addition to the advances mentioned [8] F. Xian, C. L. Hendrickson, A. G. Marshall, Anal. Chem. 2012, 84, 708. nal processing methods are still to be de- above, during its first eight years LSMB [9] R. A. Zubarev, A. Makarov, Anal. Chem. 2013, veloped. Importantly, their development has also contributed to the physical chem- 85, 5288. for SRMS may find utility in other areas istry side of tandem mass spectrometry.[27] [10] M. Scigelova, M. Hornshaw, A. Giannakopulos, of science and technology facing similar Particularly, we revealed new insights into A. Makarov, Mol. Cell. Proteomics 2011, 10, challenges. electron capture and transfer dissociation M111.009431. [11] A. Makarov, Anal. Chem. 2000, 72, 1156. fundamentals. For instance, we discovered [12] M. Gonin, US Patent 10/449328, 2003. and performed mechanistic studies of the [13] A. Verenchikov, Patent GB20100000649, 2010. Conclusions and Outlook periodic distribution of ECD/ETD product [14] D. Zajfman, Y. Rudich, I. Sagi, D. Strasser, D. ions from peptides exhibiting a tendency W. Savin, S. Goldberg, M. Rappaport, O. Heber, Int. J. Mass Spectrom. 2003, 229, 55. High-resolution MS continues to in- to form an alpha-helical secondary struc- [15] J. H. Jungmann, L. MacAleese, J. Visser, M. crease its role in molecular structural anal- ture;[50–53] completed knowledge on the J. J. Vrakking, R. M. A. Heeren, Anal. Chem. ysis.Already accepted as an important ana- role of amino acid side-chains in the ECD/ 2011, 83, 7888. lytical technique by life and environmental ETD of peptides with beta amino acids in [16] K. O. Zhurov, A. N. Kozhinov, Y. O. Tsybin, sciences researchers, it has begun to dem- the sequence;[54] suggested and computa- Anal. Chem. 2013, 85, 5311. [17] A. N. Kozhinov, T. Aushev, Y. O. Tsybin, onstrate its importance and utility for clini- tionally supported a mechanism of het- in Proc. 61st ASMS Conference on Mass cal and medical applications. Advances in erolytic N-C backbone cleavage in ECD/ Spectrometry and Allied Topics, Minneapolis, α instrumentation and signal processing, in- ETD.[55] In parallel, we applied improved MN, USA, 2013. cluding those summarized here, open new understanding of ECD/ETD fundamentals [18] Ü. A. Laskay, A. A. Lobas, K. Srzentic, M. V. Gorshkov, Y. O. Tsybin, J. Proteome Res. 2013, avenues for improved molecular analysis, for peptide structure analysis: created a 12, 5558. e.g. top–down mass spectrometry of iso- method for distinguishing N-terminal and [19] U. Laskay, A., K. Srzentic, L. Fornelli, O. Upir, lated intact monoclonal antibodies, pro- C-terminal product ions for improved pep- A. N. Kozhinov, M. Monod, Y. O. Tsybin, tein–drug complexes, or clinically relevant tide sequencing;[56] investigated the mode Chimia 2013, 67, 244. proteins for their proteoform-level analy- of action of tissue transglutaminase on pep- [20] K. Srzentic, G. Karateev, L. Fornelli, L. Levitsky, A. A. Lobas, U. A. Laskay, M. V. Gorshkov, D. [26] sis; middle–down and extended bottom–up tide deamidation and its possible impli- Ayoub, E. Dubikovskaya, Y. O. Tsybin, in Proc. proteomics of complex protein mixtures cation in Alzheimer-related beta-amyloid 62nd ASMS Conference on Mass Spectrometry for improved qualitative and quantitative peptide aggregation kinetics.[57] Improving and Allied Topics, Baltimore, USA, 2014. protein analysis; and petroleomics for a the understanding of fundamentals behind [21] Y. O. Tsybin, L. Fornelli, C. Stoermer, M. Luebeck, J. Parra, S. Nallet, F. M. Wurm, R. complete speciation of extremely complex molecular interaction with electrons, pho- Hartmer, Anal. Chem. 2011, 83, 8919. mixtures of small molecules. For instance, tons, and other molecules is indispensable [22] L. Fornelli, E. Damoc, P. M. Thomas, N. L. the methods developed in our laboratory for revealing the molecular structure-ac- Kelleher, K. Aizikov, E. Denisov, A. Makarov, for biotherapeutics, monoclonal antibod- tivity-function triad. Therefore, physical Y. O. Tsybin, Mol. Cell. Proteomics 2012, 11, ies, structural analysis can significantly chemistry naturally complements the syn- 1758. [23] L. M. Smith, N. L. Kelleher, Consortium Top– reduce the time and artifacts induced by ergy of analytical and bio-chemistry for Down Proteomics, Nature Methods 2013, 10, sample preparation in classical methods improved biomolecular structure analysis 186. which makes them useful for antibody- that constitutes the heart of LSMB inter- [24] S. Nallet, L. Fornelli, S. Schmitt, J. Parra, based drug development and characteriza- disciplinary research program. L. Baldi, Y. O. Tsybin, F. M. Wurm, New Biotechnol. 2012, 29, 471. tion. These methods can also be extended [25] L. Fornelli, D. Ayoub, K. Aizikov, A. Beck, Y. to characterization of antibody-drug con- Acknowledgements O. Tsybin, Anal. Chem. 2014, DOI: 10.1021/ jugates, which are a new generation anti- Former and current LSMB group members ac4036857. [26] L. Fornelli, A. W. Schmid, L. Grasso, H. Vogel, body-based therapeutics. and collaborators are warmly acknowledged Super-resolution mass spectrometry, Y. O. Tsybin, Chem. Eur. J. 2011, 17, 486. for all the exciting and productive interactions [27] K. O. Zhurov, L. Fornelli, M. D. Wodrich, U. implemented at LSMB for real-life FTMS over the eight years of laboratory life, since its A. Laskay, Y. O. Tsybin, Chem. Soci. Rev. 2013, data, makes its first steps through funda- inception to the current time. We thank Thermo 42, 5014. 174 CHIMIA 2014, 68, Nr. 3 Mass spectroMetry in switzerland

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