Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process

Filter Diagonalization Method-Based Mass Spectrometry for Molecular and Macromolecular Structure Analysis

Molecular and macromolecular structure analysis by high resolution and accurate mass spectrometry (MS) is indispensable for a number of fundamental and applied research areas, including health and energy domains. Comprehensive structure analysis of molecules and macromolecules present in the extremely complex samples and performed under time-constrained experimental conditions demands a substantial increase in the acquisition speed of high resolution MS data. We demonstrate here that signal processing based on the filter diagonalization method (FDM) provides the required resolution for shorter experimental transient signals in ion cyclotron resonance (ICR) MS compared to the Fourier transform (FT) processing. We thus present the development of a FDM-based MS (FDM MS) and demonstrate its implementation in ICR MS. The considered FDM MS applications are in bottom-up and top-down proteomics, metabolomics, and petroleomics

Least-Squares Fitting of Time-Domain Signals for Fourier Transform Mass Spectrometry

To advance Fourier transform mass spectrometry (FTMS)-based molecular structure analysis, corresponding development of the FTMS signal processing methods and instrumentation is required. Here, we demonstrate utility of a least-squares fitting (LSF) method for analysis of FTMS time-domain (transient) signals. We evaluate the LSF method in the analysis of single- and multiple-component experimental and simulated ion cyclotron resonance (ICR) and Orbitrap FTMS transient signals. Overall, the LSF method allows one to estimate the analytical limits of the conventional instrumentation and signal processing methods in FTMS. Particularly, LSF provides accurate information on initial phases of sinusoidal components in a given transient. For instance, the phase distribution obtained for a statistical set of experimental transients reveals the effect of the first data-point problem in FT-ICR MS. Additionally, LSF might be useful to improve the implementation of the absorption-mode FT spectral representation for FTMS applications. Finally, LSF can find utility in characterization and development of filter-diagonalization method (FDM) MS

Iterative Method for Mass Spectra Recalibration via Empirical Estimation of the Mass Calibration Function for Fourier Transform Mass Spectrometry-Based Petroleomics

We describe a mass spectra recalibration method, which enables analysis of petroleum samples with Orbitrap FTMS. In this method, the mass calibration function is estimated on the basis of mass-to-charge ratios and abundances of internal calibrants without a need for theoretical description of residual mass errors. Importantly, to maximize the estimation accuracy of the mass calibration function, an iterative approach is implemented to obtain sufficiently high number of internal calibrants covering the entire ranges of mass-to-charge ratios and abundances of interest. For petroleomic samples, the method routinely provides root-mean-square (RMS) mass accuracies at sub-ppm level and hence allows for reliable assignment of elemental compositions. Moreover, since the achieved mass accuracies are normally limited only by random errors of low-abundance analytes, the method maximizes the range of abundances of assignable species for a given signal-to-noise ratio of experimental data. Additionally, despite being initially developed for Orbitrap FTMS, the method is likewise applicable for ion cyclotron resonance FTMS

Evaluation of high-field Orbitrap Fourier transform mass spectrometer for petroleomics

The performance of the high-field Orbitrap Fourier transform mass spectrometer (FTMS) is evaluated in the context of petroleum sample analysis. Pertinent characteristics including resolving power, mass accuracy, and spectral dynamic range are tested with resin and maltene samples. Comparisons are made to the latest requirements for petroleum analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The resolving power of the high-field compact Orbitrap mass analyzer (with a commercially provided resolution setting of 480,000 at 400 m/z) is found to be sufficient for adequate characterization of the oil fractions under consideration. We demonstrate that the high-field Orbitrap FTMS may provide acceptable mass accuracy, comparable to FT-ICR MS, when an in-house developed recalibration procedure is employed. Presented data show that the high-field Orbitrap FTMS is particularly suitable for the study of lighter oil fractions, preferably with low sulfur content. Comprehensive analysis of more complex crude oil samples requires a further, at least 2-fold, increase in resolving power

Filter Diagonalization Method-Based Mass Spectrometry for Molecular and Macromolecular Structure Analysis

Molecular and macromolecular structure analysis by high resolution and accurate mass spectrometry (MS) is indispensable for a number of fundamental and applied research areas, including health and energy domains. Comprehensive structure analysis of molecules and macromolecules present in the extremely complex samples and performed under time-constrained experimental conditions demands a substantial increase in the acquisition speed of high resolution MS data. We demonstrate here that signal processing based on the filter diagonalization method (FDM) provides the required resolution for shorter experimental transient signals in ion cyclotron resonance (ICR) MS compared to the Fourier transform (FT) processing. We thus present the development of a FDM-based MS (FDM MS) and demonstrate its implementation in ICR MS. The considered FDM MS applications are in bottom-up and top-down proteomics, metabolomics, and petroleomics

Ion Trap with Narrow Aperture Detection Electrodes for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

The current paradigm in ion trap (cell) design for Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is the ion detection with wide aperture detection electrodes. Specifically, excitation and detection electrodes are typically 90A degrees wide and positioned radially at a similar distance from the ICR cell axis. Here, we demonstrate that ion detection with narrow aperture detection electrodes (NADEL) positioned radially inward of the cell's axis is feasible and advantageous for FT-ICR MS. We describe design details and performance characteristics of a 10 T FT-ICR MS equipped with a NADEL ICR cell having a pair of narrow aperture (flat) detection electrodes and a pair of standard 90A degrees excitation electrodes. Despite a smaller surface area of the detection electrodes, the sensitivity of the NADEL ICR cell is not reduced attributable to improved excite field distribution, reduced capacitance of the detection electrodes, and their closer positioning to the orbits of excited ions. The performance characteristics of the NADEL ICR cell are comparable with the state-of-the-art FT-ICR MS implementations for small molecule, peptide, protein, and petroleomics analyses. In addition, the NADEL ICR cell's design improves the flexibility of ICR cells and facilitates implementation of advanced capabilities (e.g., quadrupolar ion detection for improved mainstream applications). It also creates an intriguing opportunity for addressing the major bottleneck in FTMS-increasing its throughput via simultaneous acquisition of multiple transients or via generation of periodic non-sinusoidal transient signals

Sidebands in Fourier transform ion cyclotron resonance mass spectra

Sidebands in mass spectra are an intrinsic feature of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Appearance of the sidebands there is detrimental for the analytical performance, especially in case of complex mixtures analyzed at high resolution. Yet, the sidebands have a practical potential as well. Specifically, they can be applied for fine tuning of ICR cells and were previously employed to improve mass measurement accuracy for small molecules and atoms in fundamental physics experiments. Moreover, experimental characteristics of sidebands allow evaluating the theory of the ICR signal, which provides the metrological basis in FT-ICR MS. Here, we revisit the sidebands phenomenon in the conventional FT-ICR MS, specifically applied to macromolecules. We extend the previous reports on sidebands by examining the appearance of sidebands as functions of ICR cell trapping potentials, resolution, and number of charges for the first three harmonics of the reduced cyclotron frequency. Next, we develop an analytical model of sidebands that contributes to the existing theory of the ICR signal by showing the origin of sidebands to be the result of the broadband amplitude-phase modulation occurring in the ICR signal. Finally, we evaluate the theory of the ICR signal on the basis of the obtained experimental sidebands. Further progress in the theory of the ICR signal shall outline the way for further improvements in FT-ICR MS performance. (C) 2012 Elsevier B.V. All rights reserved

High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry with Increased Throughput for Biomolecular Analysis

A multielectrode ion cyclotron resonance (ICR) cell, herein referred to as the 4X cell, for signal detection at the quadruple frequency multiple was implemented and characterized on a commercial 10 T Fourier transform ICR mass spectrometer (FT-ICR MS). Notably, with the 4X cell operating at a 10 T magnetic field we achieved a 4-fold increase in MS acquisition rate per unit of resolving power for signal detection periods typically employed in FTMS, viz., shorter than 6 s. Effectively, the obtained resolution performance represents the limit of the standard measurement principle with dipolar signal detection and FT signal processing at an equivalent magnetic field of 40 T. In other words, the achieved resolving powers are 4 times higher than those provided by 10 T FT-ICR MS with a standard ICR cell. For example, resolving powers of 170 000 and 70 000 were obtained in magnitude-mode Fourier spectra of 768 and 192 ms apodized transient signals acquired for a singly charged fluorinated phosphazine (m/z 1422) and a 19-fold charged myoglobin (MW 16.9 kDa), respectively. In peptide analysis, the baseline-resolved isotopic fine structures were obtained with as short as 768 ms transients. In intact protein analysis, the average resolving power of 340 000 across the baseline-resolved 13C isotopic pattern of multiply charged ions of bovine serum albumin was obtained with 1.5 s transients. The dynamic range and the mass measurement accuracy of the 4X cell were found to be comparable to the ones obtained for the standard ICR cell on the same mass spectrometer. Overall, the reported results validate the advantages of signal detection at frequency multiples for increased throughput in FT-ICR MS, essential for numerous applications with time constraints, including proteomics

On the Utility of Isotopic Fine Structure Mass Spectrometry in Protein Identification

Modern mass spectrometry (MS)-based protein identification and characterization relies upon accurate mass measurements of the C-13 isotopic distributions of the enzymatically produced peptides. Interestingly, obtaining peptide elemental composition information from its isotopic fine structure mass spectrum to increase the confidence in peptide and protein identification has not yet been developed into a bottom-up proteomics-grade analytical approach. Here, we discuss the possible utility and limitations of the isotopic fine structure MS for peptide and protein identification. First, we in silica identify the peptides from the E. colt tryptic digest and show the increased confidence in peptide identification by consideration of the isotopic fine structures of these peptides as a function of mass and abundance accuracies. In the following, we demonstrate that the state-of-the-art high magnetic field Fourier transform ion cyclotron resonance (FT-ICR) MS allows a routine acquisition of the isotopic fine structure information of a number of isobaric peptide pairs, including a pair of peptides originating from E. coli. Finally, we address the practical limitation of the isotopic fine structure MS implementation in the time constraint experiments by applying an advanced signal processing technique, filter diagonalization method, to the experimental transients to overcome the resolution barrier set by the typically applied Fourier transformation. We thus demonstrate that the isotopic fine structures of peptides may indeed improve the peptide and possibly protein identification, can be produced in a routine experiment by the state-of-the-art high resolution mass spectrometers, and can be potentially obtained on a chromatographic time-scale of a typical bottom-up proteomics experiment. The latter one requires at least an order of magnitude increase in sensitivity of ion detection, which presumably can be realized using high-field Orbitrap FTMS and/or future generation of ultrahigh magnetic field FT-ICR MS equipped with harmonized ICR cells