Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer

The increasing importance of immunoglobulins G (IgGs) as biotherapeutics calls for improved structural characterization methods designed for these large (similar to 150 kDa) macromolecules. Analysis workflows have to be rapid, robust, and require minimal sample preparation. In a previous work we showed the potential of Orbitrap Fourier transform mass spectrometry (FTMS) combined with electron transfer dissociation (ETD) for the top-down investigation of an intact IgG1, resulting in-30% sequence coverage. Here, we describe a top-down analysis of two IgGs1 (adalimumab and trastuzumab) and one IgG2 (panitumumab) performed with ETD on a mass spectrometer equipped with a high-field Orbitrap mass analyzer. For the IgGs1, sequence coverage comparable to the previous results was achieved in a two-fold reduced number of summed transients, which corresponds, taken together with the significantly increased spectra acquisition rate, to-six-fold improvement in analysis time. Furthermore, we studied the influence of ion-ion interaction times on ETD product ions for IgGs1, and the differences in fragmentation behavior between IgGs1 and IgG2, which present structural differences. Overall, these results reinforce the hypothesis that gas phase dissociation using both energy threshold-based and radical driven ion activations is directed to specific regions of the polypeptide chains mostly by the location of disulfide bonds. Significance of the study: Compared with our previous report, the results presented herein demonstrate the power of technological advances of the next generation Orbitrap (TM) platform, including the use of a high-field compact (i.e., D20) Orbitrap mass analyzer, and a dedicated manipulation strategy for large protein ions (via their trapping in the HCD collision cell along with reduction of the pressure in the cell). Notably, these important developments became recently commercially available in the top-end Orbitrap platforms under the name of "Protein Mode". Furthermore, we continued exploring the advantages offered by the summation (averaging) of transients (time-domain data) for improving the signal-to-noise ratio of top-down mass spectra. Finally, for the first time we report the application of the hybrid ion activation technique that combines electron transfer dissociation and higher energy collisional dissociation, known as EThcD, on intact monoclonal antibodies. Under these specific instrumental parameters, EThcD produces a partially complementary fragmentation pattern compared to ETD, increasing the overall sequence coverage especially at the protein termini. (C) 2017 Elsevier B.V. All rights reserved.

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

Luca Fornelli, Yury Tsybin

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. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019620, 1758-1767, 2012.

Amer Soc Biochemistry Molecular Biology Inc2012

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry

Yury Tsybin

An electron injection system based on an indirectly heated ring-shaped dispenser cathode was developed and installed in a 7 T Fourier transform ICR (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissocn. (ECD) to be carried out by a hollow electron beam coaxial with the ICR trap. IR multiphoton dissocn. (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the center of the dispenser cathode. Electron and photon irradn. times of the order of 100 ms are required for efficient ECD and IRMPD, resp. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, resp.), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR exptl. sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MSn) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the anal. of protein digests sepd. by liq. chromatog. prior to FTICRMS. Also, ion activation by either electron or laser irradn. prior to, as well as after, dissocn. by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap. [on SciFinder (R)]

2003

Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry

Luca Fornelli, Sophie Nallet, Julien Parra, Yury Tsybin, Florian Maria Wurm

Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

2011