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

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. 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) 1The abbreviations used are:ETDelectron transfer dissociationECDelectron capture dissociationCIDcollision-induced dissociationFTFourier transformFTMSFourier transform mass spectrometryIgGimmunoglobulin GMSmass spectrometryMS/MStandem mass spectrometryLCliquid chromatographyPTMpost-translational modificationSNRsignal-to-noise ratioFT-ICR MSFourier transform ion cyclotron resonance mass spectrometryMWmolecular weightqTOF MSquadrupole time-of-flight mass spectrometryLTQlinear trap quadrupole (linear ion trap)pyroGlupyroglutamic acid. 1The abbreviations used are:ETDelectron transfer dissociationECDelectron capture dissociationCIDcollision-induced dissociationFTFourier transformFTMSFourier transform mass spectrometryIgGimmunoglobulin GMSmass spectrometryMS/MStandem mass spectrometryLCliquid chromatographyPTMpost-translational modificationSNRsignal-to-noise ratioFT-ICR MSFourier transform ion cyclotron resonance mass spectrometryMWmolecular weightqTOF MSquadrupole time-of-flight mass spectrometryLTQlinear trap quadrupole (linear ion trap)pyroGlupyroglutamic acid. 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Chafey P. Camoin L. Nilges M. Nassif X. Dumenil G. Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.Science. 2011; 331: 778-782Crossref PubMed Scopus (143) Google Scholar). 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 (5Schluter H. Apweiler R. Holzhutter H.G. Jungblut P.R. Finding one's way in proteomics: a protein species nomenclature.Chem. Cent. J. 2009; 3: 11Crossref PubMed Scopus (198) Google Scholar). 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 (6Zhang 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.Biochemistry. 2011; 50: 6081-6092Crossref PubMed Scopus (29) Google Scholar), 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 (7Nikolaev E.N. Boldin I.A. Jertz R. Baykut G. Initial experimental characterization of a new ultra-high resolution FTICR cell with dynamic harmonization.J. Am. Soc. Mass Spectr. 2011; 22: 1125-1133Crossref PubMed Scopus (124) Google Scholar, 8Tsybin 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.J. Mass Spectrom. 2004; 39: 719-729Crossref PubMed Scopus (32) Google Scholar). However, this solution has been shown to have some limitations in the analysis of large proteins (9Patrie 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.Mol. Cell. Proteomics. 2006; 5: 14-25Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The main issue, as described by Compton et al. (10Compton P.D. Zamdborg L. Thomas P.M. Kelleher N.L. On the scalability and requirements of whole protein mass spectrometry.Anal. Chem. 2011; 83: 6868-6874Crossref PubMed Scopus (142) Google Scholar), is that the SNR in Fourier transform mass spectrometry (FTMS) is inversely proportional to the width of the isotopic and charge state distributions (11Scigelova M. Hornshaw M. Giannakopulos A. Makarov A. Fourier transform mass spectrometry.Mol. Cell. Proteomics. 2011; 10 (O111.009431)Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), which both increase as a function of MW. 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The ETD mass from scans with fragmentation of different IgG ions and and for the to and and to for the the the was observed to be also in acid as in in The number of ETD of the charge of IgG the of the and of the resulting in potential to sequence the both of in ETD not result in of the are in the for the and 10 for the are in of the is that the of the of both variable and has been with of the by the identification of information about the and the and/or However, the of the the was not of ions from top-down MS of sequence of sequence of sequence scans, in the present on intact human with for the and and for the scans, in the present on intact human with for the and and for the by the of the Orbitrap FTMS in a previous on an intact with for the and for the from on and human with for the and for the in the present on intact human with for the and and for the by the of the Orbitrap FTMS in a previous Y.O. L. C. M. J. S. R. analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry.Anal. Chem. 2011; 83: PubMed Scopus Google Scholar) on an intact with for the and for the from Makarov Zhang Mass and top-down analysis of intact monoclonal antibodies on a hybrid quadrupole ion mass Am. Soc. Mass Spectrom. 2009; PubMed Scopus Google on and human with for the and for the in a new The ion ions from to charge which might new dissociation was employed in to the ETD sequence ETD of ions from the and from the and The number of to sequence to the ETD results with the the of the a lower number of whereas the sequence coverage of the between the and and and the of the is The in fragmentation is in the of ions the number of ions information on the and was in ETD with the whereas for ETD with the for the for the the number from for the former to for the latter ion the SNR both mass the observed in sequence coverage the are to the of different ETD ions for the experimental the by Orbitrap ETD MS/MS for the structural analysis of intact and IgG, a with previous ETD MS/MS on a MS in Y.O. L. C. M. J. S. R. analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry.Anal. Chem. 2011; 83: PubMed Scopus Google and MS/MS on an Orbitrap FTMS Makarov Zhang Mass and top-down analysis of intact monoclonal antibodies on a hybrid quadrupole ion mass Am. Soc. Mass Spectrom. 2009; PubMed Scopus Google Scholar) can be that experiments on and IgG of a different not ETD the human different only in the variable as to the and the increase in sequence coverage provided by ETD on Orbitrap FTMS is we the results of and ETD Orbitrap FTMS the number of to sequence of the of sequence coverage on the number of scans has to be Given their and and disulfide and on and their high molecular weight the characterization of intact a for MS The state-of-the-art high resolution Orbitrap FTMS allows intact IgG charge state in a to and fragmentation by both and higher and dissociation also the of multiply charged ions present in a a top-down MS approach for the structural analysis of intact Here, we have that time-domain signal averaging from a number of LC-MS/MS experiments before signal and the of Orbitrap top-down that state-of-the-art top-down proteomics of 10 to proteins is with on-line the analysis of intact proteins than is protein by for an on-line LC-MS/MS approach because is than the approach in of the of to be as well as its for automated quality the of the top-down proteomics is to of protein that proteins as large as 150 to an increase in MS sensitivity and speed of high resolution LC-MS/MS be required for protein the the observed increase in SNR as with the of the number of we a to in SNR from a single LC-MS/MS experiment to the mass to the number of we not only scans, but we also used a large ion a the demonstrate a 2-fold higher sequence coverage than was on a similar human in a prior ETD for top-down MS analysis of we the of to on the IgG that structural between IgG in ETD MS/MS be to similar IgG information about the ETD for a particular IgG sequence or higher an of information on the ETD The of the results for ETD-based top-down MS is primarily in the improved of the employed experimental that increase the of the and an to be its previous the also the in of the mass resolving The large number of with Orbitrap FTMS a high of the results described similar to we not to sequence of the of or the of the because of a of and the of the and of the IgG in the which the almost complete sequence coverage for the in the and are by ion before and ETD IgG the of disulfide and the number of ion in the of a Orbitrap mass or Orbitrap FTMS instrument or improved ion transfer and to increase the speed of analysis and the number of scans required for top-down analysis. are to for with the analysis. with

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