Christopher Mullen

Novel Parallelized Quadrupole/Linear Ion Trap/Orbitrap Tribrid Mass Spectrometer Improving Proteome Coverage and Peptide Identification Rates

Proteome coverage and peptide identification rates have historically advanced in line with improvements to the detection limits and acquisition rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid, the acquisition rate has been limited primarily by the duration of the ion accumulation and analysis steps. It is shown here that the spectral acquisition rate can be significantly improved through extensive parallelization of the acquisition process using a novel mass spectrometer incorporating quadrupole, Orbitrap, and linear trap analyzers. Further, these improvements to the acquisition rate continue to enhance proteome coverage and general experimental throughput.

Benchmarking Multiple Fragmentation Methods on an Orbitrap Fusion for Top-down Phospho-Proteoform Characterization

Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms. Data are available via ProteomeXchange with identifier PXD001845.

Front-End Electron Transfer Dissociation: A New Ionization Source

Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion-ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression

Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such “trivalent hypermethylation” (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.

Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation.

AbstractElectron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately 3-fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase. Graphical Abstractᅟ

A Calibration Routine for Efficient ETD in Large-Scale Proteomics

AbstractElectron transfer dissociation (ETD) has been broadly adopted and is now available on a variety of commercial mass spectrometers. Unlike collisional activation techniques, optimal performance of ETD requires considerable user knowledge and input. ETD reaction duration is one key parameter that can greatly influence spectral quality and overall experiment outcome. We describe a calibration routine that determines the correct number of reagent anions necessary to reach a defined ETD reaction rate. Implementation of this automated calibration routine on two hybrid Orbitrap platforms illustrate considerable advantages, namely, increased product ion yield with concomitant reduction in scan rates netting up to 75% more unique peptide identifications in a shotgun experiment. Graphical Abstractᅟ

LASER-IONIZATION MASS SPECTROMETRY OF EXPLOSIVES AND CHEMICAL WARFARE SIMULANTS

The objective of the present study was to better understand the photophysics of explosives and chemical warfare simulants in order to develop better performing analytical tools. Photoionization mass spectra were taken using three optical schemes. The first was resonance-enhanced multiphoton ionization (REMPI) using few-ns duration 248 or 266 nm laser pulses. The second scheme was non-resonant multiphoton ionization (MPI) using 100 fs duration laser pulses at wavelengths between 325 and 795. The third approach was single photon ionization (SPI) using few-ns duration 118 nm laser pulses. For all the molecules investigated, mass spectra resulting exposure to ns-duration 248 or 266 nm laser pulses consisted of only low molecular weight fragments. Using fs-duration laser pulses produced more complicated, potentially analyzable, fragmentation patterns, usually with some parent peak. Single photon ionization gave the best results, with mass spectra consisting of almost only parent peak, except for the case of TATP.

Accurate sequence analysis of a monoclonal antibody by top-down and middle-down Orbitrap mass spectrometry applying multiple ion activation techniques

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.

Implementation of Fragment Ion Protection (FIP) during Ultraviolet Photodissociation (UVPD) Mass Spectrometry

Ultraviolet photodissociation (UVPD) is a nonselective activation method in which both precursor and fragment ions may absorb photons and dissociate. Photoactivation of fragment ions may result in secondary or multiple generations of dissociation, which decreases the signal-to-noise ratio (S/N) of larger fragment ions owing to the prevalent subdivision of the ion current into many smaller, often less informative, fragment ions. Here we report the use of dipolar excitation waveforms to displace fragment ions out of the laser beam path, thus alleviating the extent of secondary dissociation during 193 nm UVPD. This fragment ion protection (FIP) strategy increases S/N of larger fragment ions and improves the sequence coverage obtained for proteins via retaining information deeper into the midsection of protein sequences.

Detection of Explosive and Explosive Related Compounds (ERCs) Using Ultrafast Laser Photoionization Time of Flight Mass Spectrometry

Ultrafast laser photoionization time of flight mass spectrometry has been applied to the detection of explosives and explosive related compounds. The wavelength dependence of the photoionization event and the sensitivity of the technique are discussed.