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Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS*

Open AccessPublished:September 10, 2012DOI:https://doi.org/10.1074/mcp.M112.019620
      The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure–function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.
      Top-down mass spectrometry (MS)
      The abbreviations used are:
      ETD
      electron transfer dissociation
      ECD
      electron capture dissociation
      CID
      collision-induced dissociation
      FT
      Fourier transform
      FTMS
      Fourier transform mass spectrometry
      IgG
      immunoglobulin G
      MS
      mass spectrometry
      MS/MS
      tandem mass spectrometry
      LC
      liquid chromatography
      PTM
      post-translational modification
      SNR
      signal-to-noise ratio
      FT-ICR MS
      Fourier transform ion cyclotron resonance mass spectrometry
      MW
      molecular weight
      qTOF MS
      quadrupole time-of-flight mass spectrometry
      LTQ
      linear trap quadrupole (linear ion trap)
      pyroGlu
      pyroglutamic acid.
      1The abbreviations used are:ETD
      electron transfer dissociation
      ECD
      electron capture dissociation
      CID
      collision-induced dissociation
      FT
      Fourier transform
      FTMS
      Fourier transform mass spectrometry
      IgG
      immunoglobulin G
      MS
      mass spectrometry
      MS/MS
      tandem mass spectrometry
      LC
      liquid chromatography
      PTM
      post-translational modification
      SNR
      signal-to-noise ratio
      FT-ICR MS
      Fourier transform ion cyclotron resonance mass spectrometry
      MW
      molecular weight
      qTOF MS
      quadrupole time-of-flight mass spectrometry
      LTQ
      linear trap quadrupole (linear ion trap)
      pyroGlu
      pyroglutamic acid.
      (
      • Cui W.
      • Rohrs H.W.
      • Gross M.L.
      Top-down mass spectrometry: recent developments, applications and perspectives.
      ,
      • Kellie J.F.
      • Tran J.C.
      • Lee J.E.
      • Ahlf D.R.
      • Thomas H.M.
      • Ntai I.
      • Catherman A.D.
      • Durbin K.R.
      • Zamdborg L.
      • Vellaichamy A.
      • Thomas P.M.
      • Kelleher N.L.
      The emerging process of top down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput.
      ,
      • Tipton J.D.
      • Tran J.C.
      • Catherman A.D.
      • Ahlf D.R.
      • Durbin K.R.
      • Kelleher N.L.
      Analysis of intact protein isoforms by mass spectrometry.
      ) has continued to demonstrate its particular advantages over traditionally employed bottom-up MS strategies (
      • Chamot-Rooke J.
      • Mikaty G.
      • Malosse C.
      • Soyer M.
      • Dumont A.
      • Gault J.
      • Imhaus A.F.
      • Martin P.
      • Trellet M.
      • Clary G.
      • Chafey P.
      • Camoin L.
      • Nilges M.
      • Nassif X.
      • Dumenil G.
      Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.
      ). Specifically, top-down MS allows the characterization of specific protein isoforms originating from the alternative splicing of mRNA that code single nucleotide polymorphisms and/or post-translational modifications (PTMs) of protein species (
      • Schluter H.
      • Apweiler R.
      • Holzhutter H.G.
      • Jungblut P.R.
      Finding one's way in proteomics: a protein species nomenclature.
      ). Intact protein molecular weight (MW) determination and subsequent gas-phase fragmentation of selected multiply charged protein ions (referred to as tandem MS or MS/MS) theoretically might result in complete protein sequence coverage and precise assignment of the type and position of PTMs, amino acid substitutions, and C- or N-terminal truncations (
      • Zhang J.
      • Zhang H.
      • Ayaz-Guner S.
      • Chen Y.C.
      • Dong X.
      • Xu Q.
      • Ge Y.
      Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T.
      ), whereas the bottom-up MS approach allows only the identification of a certain protein family when few or redundant peptides are found for a particular protein isoform. At a practical level, however, top-down MS-based proteomics struggles not only with the single- or multi-dimensional separation of undigested proteins, which demonstrates lower reproducibility and repeatability than for peptides, but also with technical limitations present in even state-of-the-art mass spectrometers. The outcome of a top-down MS experiment depends indeed on the balance between the applied resolution of the mass spectrometer and its sensitivity. The former is required for unambiguous assignment of ion isotopic clusters in both survey and MS/MS scans, whereas the latter is ultimately dependent on the scan speed of the mass analyzer, which determines the number of scans that can be accumulated for a given analyte ion on the liquid chromatography (LC) timescale to enhance the resulting signal-to-noise ratio (SNR). Until recently, the instrument of choice for top-down MS has been the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, primarily because of its superior resolving power and the availability of electron capture dissociation for the efficient MS/MS of large biomolecules (
      • Nikolaev E.N.
      • Boldin I.A.
      • Jertz R.
      • Baykut G.
      Initial experimental characterization of a new ultra-high resolution FTICR cell with dynamic harmonization.
      ,
      • Tsybin Y.O.
      • Ramstrom M.
      • Witt M.
      • Baykut G.
      • Hakansson P.
      Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry.
      ). However, this solution has been shown to have some limitations in the analysis of large proteins (
      • Patrie S.M.
      • Ferguson J.T.
      • Robinson D.E.
      • Whipple D.
      • Rother M.
      • Metcalf W.W.
      • Kelleher N.L.
      Top down mass spectrometry of < 60-kDa proteins from Methanosarcina acetivorans using quadrupole FTMS with automated octopole collisionally activated dissociation.
      ). The main issue, as described by Compton et al. (
      • Compton P.D.
      • Zamdborg L.
      • Thomas P.M.
      • Kelleher N.L.
      On the scalability and requirements of whole protein mass spectrometry.
      ), is that the SNR in Fourier transform mass spectrometry (FTMS) is inversely proportional to the width of the isotopic and charge state distributions (
      • Scigelova M.
      • Hornshaw M.
      • Giannakopulos A.
      • Makarov A.
      Fourier transform mass spectrometry.
      ), which both increase as a function of MW. Particularly, the SNR dramatically decreases with MW under standard on-line LC-MS/MS operating conditions if isotopic resolution is required. It is noteworthy that such SNR reduction can affect not only intact mass measurements, but also the subsequent MS/MS performance.
      The most widely employed solution for improving top-down analysis is thus a substantial reduction of the protein mixture complexity, for example, through off-line sample prefractionation (
      • Tran J.C.
      • Doucette A.A.
      Multiplexed size separation of intact proteins in solution phase for mass spectrometry.
      ). Furthermore, when the MW exceeds 100 kDa, proteins are often analyzed via direct infusion after off-line purification of the single isoform or species of interest (
      • Ge Y.
      • Rybakova I.N.
      • Xu Q.
      • Moss R.L.
      Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state.
      ). Overall, these strategies aim to improve the quality of mass spectra, specifically their SNR, by increasing the number of scans dedicated to each selected isoform or species. However, off-line intact protein analysis has limitations, including sample degradation and modification (e.g., oxidation during long off-line measurements and sample storage). The time required for multistep LC-based protein purification can also be substantial.
      Electron capture dissociation (ECD) (
      • Zubarev R.A.
      • Kelleher N.L.
      • McLafferty F.W.
      Electron capture dissociation of multiply charged protein cations. A nonergodic process.
      ,
      • Zubarev R.A.
      Reactions of polypeptide ions with electrons in the gas phase.
      ) and electron transfer dissociation (ETD) (
      • Syka J.E.P.
      • Coon J.J.
      • Schroeder M.J.
      • Shabanowitz J.
      • Hunt D.F.
      Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.
      ) are ion activation techniques that allow polypeptide fragmentation with reduced PTM losses (
      • Mirgorodskaya E.
      • Roepstorff P.
      • Zubarev R.A.
      Localization of O-glycosylation sites in peptides by electron capture dissociation in a Fourier transform mass spectrometer.
      ,
      • Stensballe A.
      • Jensen O.N.
      • Olsen J.V.
      • Haselmann K.F.
      • Zubarev R.A.
      Electron capture dissociation of singly and multiply phosphorylated peptides.
      ). Nevertheless, ECD and ETD generally provide larger sequence coverage for intact proteins than slow-heating activation methods such as collision induced dissociation (CID) and infrared multiple photon dissociation (
      • Molina H.
      • Matthiesen R.
      • Kandasamy K.
      • Pandey A.
      Comprehensive comparison of collision induced dissociation and electron transfer dissociation.
      ,
      • Santos L.F.A.
      • Eberlin M.N.
      • Gozzo F.C.
      IRMPD and ECD fragmentation of intermolecular cross-linked peptides.
      ). Furthermore, ECD and ETD are known to cleave disulfide bonds, a fundamental feature for the analysis of proteins in their native state (i.e., without cysteine reduction and alkylation) (
      • Zubarev R.A.
      • Kruger N.A.
      • Fridriksson E.K.
      • Lewis M.A.
      • Horn D.M.
      • Carpenter B.K.
      • McLafferty F.W.
      Electron capture dissociation of gaseous multiply-charged proteins is favored at disulfide bonds and other sites of high hydrogen atom affinity.
      ,
      • Zubarev R.A.
      • Horn D.M.
      • Fridriksson E.K.
      • Kelleher N.L.
      • Kruger N.A.
      • Lewis M.A.
      • Carpenter B.K.
      • McLafferty F.W.
      Electron capture dissociation for structural characterization of multiply charged protein cations.
      ,
      • Wu S.L.
      • Jiang H.T.
      • Hancock W.S.
      • Karger B.L.
      Identification of the unpaired cysteine status and complete mapping of the 17 disulfides of recombinant tissue plasminogen activator using LC-MS with electron transfer dissociation/collision induced dissociation.
      ).
      The structural analysis of high MW intact proteins with MS has garnered much recent attention in the literature (
      • Valeja S.G.
      • Kaiser N.K.
      • Xian F.
      • Hendrickson C.L.
      • Rouse J.C.
      • Marshall A.G.
      Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry.
      ,
      • Tsybin Y.O.
      • Fornelli L.
      • Stoermer C.
      • Luebeck M.
      • Parra J.
      • Nallet S.
      • Wurm F.M.
      • Hartmer R.
      Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry.
      ), mainly because of the improved capabilities offered by rapidly developing sample preparation, protein separation, and mass spectrometric methods and techniques. Immunoglobulin G (IgG) proteins are antibodies with an MW of about 150 kDa that are composed of two identical sets of light and glycosylated heavy chains with both intra- and intermolecular disulfide bridges (Fig. 1) (
      • Zhang Z.Q.
      • Pan H.
      • Chen X.Y.
      Mass spectrometry for structural characterization of therapeutic antibodies.
      ). IgGs represent an attractive target for structural analysis method development, given their high importance as biotherapeutics (
      • Sheridan C.
      Fresh from the biologic pipeline-2009.
      ). A unit-mass resolution mass spectrum demonstrating an isotopic distribution of an isolated charge state of a 148 kDa IgG1 has been recently achieved with FT-ICR MS equipped with 9.4 T superconducting magnet and a statically harmonized ICR cell (
      • Valeja S.G.
      • Kaiser N.K.
      • Xian F.
      • Hendrickson C.L.
      • Rouse J.C.
      • Marshall A.G.
      Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry.
      ). However, further analytical improvements are needed to achieve routine and reproducible MS operation at the required level of resolution and sensitivity.
      Figure thumbnail gr1
      Fig. 1Schematic representation of IgG1. Two identical light (blue) and two identical heavy (fucsia) chains form the intact IgG. The light chain is composed of a variable domain (VL) and a constant domain (CL), whereas the heavy chain comprises one variable domain (VH) and three constant domains (CH1–3). Each domain contains an intramolecular disulfide bridge (in red); intermolecular disulfide bridges link the heavy chains to each other (two bonds) and each heavy chain to one light chain (one bond). Each heavy chain includes an N-glycosylation site (located at Asn297; here, a G0F/G0F glycosylation is shown).
      Fragmentation of intact antibodies in the gas phase following the top-down MS approach has been previously attempted without precursor ion charge state isolation by means of nozzle-skimmer CID on a linear trap quadrupole (LTQ)-Orbitrap™ (
      • Zhang Z.
      • Shah B.
      Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry.
      ,
      • Bondarenko P.V.
      • Second T.P.
      • Zabrouskov V.
      • Makarov A.A.
      • Zhang Z.Q.
      Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer.
      ) and with precursor ion isolation via ETD on a high resolution quadrupole time-of-flight (qTOF) mass spectrometer (
      • Tsybin Y.O.
      • Fornelli L.
      • Stoermer C.
      • Luebeck M.
      • Parra J.
      • Nallet S.
      • Wurm F.M.
      • Hartmer R.
      Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry.
      ). Relative to the results previously obtained with slow-heating MS/MS methods, the ETD qTOF MS/MS demonstrated substantially higher sequence coverage, reaching 15% for human and 21% for murine IgGs. Important for future top-down proteomics development for complex protein mixtures, the ETD qTOF MS/MS results were obtained on the LC timescale. To increase the sequence coverage and confidence in product ion assignment, a substantial increase in SNR was achieved by averaging MS/MS data from up to 10 identical LC-MS/MS experiments. The high complexity of the product ion population reduced the effective resolution to about 30,000, presumably limiting the assignment of overlapping high charge state product ions in the 1000–2000 m/z range. Even higher peak complexity was observed in the region of charge reduced species and complementary heavy product ions, above 3000 m/z. Finally, numerous disulfide bonds drastically reduced MS/MS efficiency in the disulfide bond-protected regions.
      Here we demonstrate that ETD-enabled hybrid linear ion trap Orbitrap FTMS allows us to further improve the top-down ETD-based LC-MS/MS of monoclonal antibodies, introduced earlier for TOF-based MS. To fully take advantage of the high resolving power of Orbitrap MS/MS for increasing both the number of assigned product ions and the confidence of the assignments, maintaining an LC-MS/MS setup useful in a general proteomics workflow for protein desalting and separation, we averaged time-domain transients (derived from separated LC-MS/MS runs) before Fourier transform signal processing.

      DISCUSSION

      Given their multimeric structure comprising two heavy and two light chains, complex intra- and intermolecular disulfide connectivity, and heterogeneous glycosylation on heavy chains and their high molecular weight (∼150 kDa), the full characterization of intact IgGs represents a difficult challenge for any MS platform. The state-of-the-art high resolution Orbitrap FTMS allows LC-timescale-compatible intact IgG ionization, charge state selection in a range up to 4000 m/z, and fragmentation by both slow-heating methods (e.g., CID and higher energy collisional dissociation) and radical chemistry-based dissociation (e.g., ETD). It also supports the detection of isotopically resolved, multiply charged product ions present in a complex mixture, enabling a top-down MS approach for the structural analysis of intact IgGs. Here, we have demonstrated that time-domain transient signal averaging from a number of consecutive LC-MS/MS experiments before FT signal processing and peak picking significantly improves the performance of Orbitrap FTMS-based top-down MS. We note that although state-of-the-art top-down proteomics of 10 to 50 kDa proteins is already performed with on-line nanoLCMS, the analysis of intact proteins heavier than 50 kDa is often performed after off-line LC-based protein purification followed by off-line nanospray-MS. We opted for an on-line LC-MS/MS approach here because it is more universal than the off-line approach in terms of the types of samples to be addressed, as well as its suitability for automated quality control workflows. Indeed, the near-future goal of the top-down proteomics is to perform analyses of complex protein mixtures that contain heavy proteins as large as 150 to 200 kDa. With an increase in MS sensitivity and speed of high resolution data acquisition, substantially fewer LC-MS/MS runs will be required for protein characterization.
      In the current work, the observed increase in SNR rose as expected with the square root of the number of scans; specifically, we estimated a roughly 4-fold to 8-fold gain in SNR from a single LC-MS/MS experiment to the final mass spectra. Furthermore, to maximize the number of assigned unique cleavage sites, we not only averaged 1000 scans, but we also used a large (600 m/z) precursor ion isolation window. As a result, the data presented here demonstrate a 2-fold higher sequence coverage than was obtained on a similar human IgG1 in a prior ETD implementation for top-down MS analysis of IgGs. In addition, we report the partial cyclization of Glu1 to pyroGlu1 on the IgG heavy chain. We note that significant structural differences between IgG variants considered in Table I certainly influence ETD MS/MS performance. Further work should be dedicated to comparing similar IgG subclasses. Also, information about the ETD preference for a particular IgG sequence or higher order structure provides an additional source of information on the ETD fundamentals.
      The novelty of the results presented here for ETD-based top-down MS is primarily in the substantially improved analytical characteristics of the employed experimental set-up that increase the overall capabilities of the method and allow an important step to be taken toward its routine application. Although previous work demonstrated the proof-of-principle implementation, it also demonstrated the corresponding limitations, specifically in terms of the mass resolving power. The large number of laboratories equipped nowadays with high-resolution Orbitrap FTMS instrumentation suggests a high impact of the results described here. Nevertheless, similar to previously reported results, we were not able to sequence long portions of the constant domains of light chains or the entire central portion of the heavy chain, presumably because of a combination of disulfide-bond networks and the partial retention of the secondary and tertiary structures of the IgG in the gas phase, which would explain the almost complete sequence coverage obtained for the flexible, unstructured loops connecting consecutive domains in the light and heavy chains (see Fig. 5). We are currently addressing these issues by implementing ion activation before and after ETD reaction, unfolding IgG via the partial reduction of disulfide bonds, and increasing the number of protonated sites via precursor ion supercharging. Future improvements in instrumentation—for example, the use of a compact high-field Orbitrap mass analyzer or Orbitrap Elite FTMS instrument or improved ion transfer and trapping—are expected to increase substantially the speed of analysis and reduce the number of scans required for top-down analysis.

      Acknowledgments

      We are grateful to Anton Kozhinov for assistance with the data analysis.

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