Abstract
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.
Highlights
The abbreviations used are: ETD, electron transfer dissociation; Electron capture dissociation (ECD), electron capture dissociation; collision induced dissociation (CID), collision-induced dissociation; FT, Fourier transform; FTMS, Fourier transform mass spectrometry; Immunoglobulin G (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; pyroGlu, pyroglutamic acid
The high resolution employed in this work profoundly contributed to the identification of IgG backbone cleavage sites, it must be stressed that the increase in SNR achieved with the addition of transients taken from different LC-MS/MS experiments is currently the most important factor for the successful analysis of populations of large, multiply charged product ions via FTMS
Most of the assigned cleavage sites are located in the disulfide-bond-free regions, 22 backbone a Measurements performed in the present study on intact human IgG1, kappa, with c-ions considered for the N-terminus and z●- and y-ions considered for the C-terminus. b Values calculated by combining the backbone cleavage sites of the two Orbitrap FTMS experiments. c Measurements performed in a previous work (Ref. 25) on an intact IgG1, kappa, with c-ions considered for the N-terminus and z●-ions considered for the C-terminus. d Data derived from Ref. [29]; measurements performed on reduced and alkylated human IgG2, with b-ions considered for the N-terminus and y-ions considered for the C-terminus
Summary
Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS*□S. 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. 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. 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
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