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