Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer

Kline, Jake; Mullen, Christopher; Durbin, Kenneth R.; Oates, Ryan; Huguet, Romain; Syka, John E. P.; Fornelli, Luca

doi:10.26434/chemrxiv.14036810.v1

Cited by 5 publications

(7 citation statements)

References 52 publications

(69 reference statements)

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“…5C) [28]. The major challenge of TDP is that its gas-phase dissociation produces overlapping and low signal-tonoise (S/N) fragments with increasing molecular weight [94]. Therefore, to better characterization of Ub chains by TDP, it's important to optimize the instrumental parameters [95].…”

Section: Insights Into the Ubiquitinated Proteins At The Level Of Arc...mentioning

confidence: 99%

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Sun

Zhang

2022

Cell Biosci

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Ubiquitination is a versatile post-translational modification (PTM), which regulates diverse fundamental features of protein substrates, including stability, activity, and localization. Unsurprisingly, dysregulation of the complex interaction between ubiquitination and deubiquitination leads to many pathologies, such as cancer and neurodegenerative diseases. The versatility of ubiquitination is a result of the complexity of ubiquitin (Ub) conjugates, ranging from a single Ub monomer to Ub polymers with different length and linkage types. To further understand the molecular mechanism of ubiquitination signaling, innovative strategies are needed to characterize the ubiquitination sites, the linkage type, and the length of Ub chain. With advances in chemical biology tools, computational methodologies, and mass spectrometry, protein ubiquitination sites and their Ub chain architecture have been extensively revealed. The obtained information on protein ubiquitination helps to crack the molecular mechanism of ubiquitination in numerous pathologies. In this review, we summarize the recent advances in protein ubiquitination analysis to gain updated knowledge in this field. In addition, the current and future challenges and barriers are also reviewed and discussed.

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Section: Insights Into the Ubiquitinated Proteins At The Level Of Arc...mentioning

confidence: 99%

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Sun

Zhang

2022

Cell Biosci

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“…Kline et al used PTCR to minimize the signal overlap and identify more UVPD and EThcD fragments. 20 However, even with no improvements to the signal overlap, UVPD contributes to unique bond cleavages, making it valuable in the multimodal approach.…”

Section: Contribution Of Individual Ms/ms Fragmentation Modes To the ...mentioning

confidence: 99%

Effective Amino Acid Sequencing of Intact Filgrastim by Multimodal Mass Spectrometry with Topdownr

Babović

Shliaha

Gibb

et al. 2022

J. Am. Soc. Mass Spectrom.

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Therapeutic proteins, known as biologicals, are an important and growing class of drugs for treatment of a series of human ailments. Amino acid sequence variants of therapeutic proteins can affect their safety and efficacy. Top-down mass spectrometry is well suited for the sequence analysis of intact therapeutic proteins. Fine-tuning of tandem mass spectrometry (MS/ MS) fragmentation conditions is essential for maximizing the amino acid sequence coverage but is often time-consuming. We used topdownr, an automated and integrated multimodal approach to systematically assess high mass accuracy MS/MS fragmentation parameters to characterize filgrastim, a 19 kDa recombinant human granulocyte colony-stimulating factor used in treating neutropenia. A total of 276 different MS/MS conditions were systematically tested, including the following parameters: protein charge state, HCD and CID collision energy, ETD reaction time, ETD supplemental activation, and UVPD activation time. Stringent and accurate evaluation and annotation of the MS/MS data was achieved by requiring a fragment ion mass error of 5 ppm, considering reproducible N-and C-terminal fragment ions only, and excluding internal fragment ion assignments. We report the first EThcD and UVPD MS/MS analysis of intact filgrastim, and these two techniques combined resulted in 98% amino acid sequence coverage. By combining all tested fragmentation modes, we obtained near-complete amino acid sequence coverage (99.4%) of intact filgrastim.

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“…6,14,35−43 Spectral congestion has been reduced following activation of intact proteins by using proton-transfer charge reduction (PTCR) reactions to lower the charge states of fragment ions and disperse them over a broad m/z range. 6,14,35,39,43 In recent studies, this process decreased spectral congestion and improved sequence coverage of proteins after UVPD 14 and ETD. 39 Ion charge states can be further manipulated through an auxiliary method known as ion parking, inhibiting the rate of ion−ion reactions by application of ion-selective waveforms.…”

Section: ■ Introductionmentioning

confidence: 99%

Impact of Internal Fragments on Top-Down Analysis of Intact Proteins by 193 nm UVPD

et al. 2022

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193 nm ultraviolet photodissociation (UVPD) allows high sequence coverage to be obtained for intact proteins using terminal fragments alone. However, internal fragments, those that contain neither N- nor C- terminus, are typically ignored, neglecting their potential to bolster characterization of intact proteins. Here, we explore internal fragments generated by 193 nm UVPD for proteins ranging in size from 17–47 kDa and using the ClipsMS algorithm to facilitate searches for internal fragments. Internal fragments were only retained if identified in multiple replicates in order to reduce spurious assignments and to explore the reproducibility of internal fragments generated by UVPD. Inclusion of internal fragment improved sequence coverage by an average of 18% and 32% for UVPD and HCD, respectively, across all proteins and charge states studied. However, only an average of 18% of UVPD internal fragments were identified in two out of three replicates relative to the average number identified across all replicates for all proteins studied. Conversely, for HCD, an average of 63% of internal fragments were retained across replicates. These trends reflect an increased risk of false-positive identifications and a need for caution when considering internal fragments for UVPD. Additionally, proton-transfer charge reduction (PTCR) reactions were performed following UVPD or HCD to assess the impact on internal fragment identifications, allowing up to 20% more fragment ions to be retained across multiple replicates. At this time, it is difficult to recommend the inclusion of the internal fragment when searching UVPD spectra without further work to develop strategies for reducing the possibilities of false-positive identifications. All mass spectra are available in the public repository jPOST with the accession number JPST001885.

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Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer

Abstract: We are submitting a manuscript describing the application of proton transfer charge reduction to increase the sequence coverage for proteins > 30 kDa using Orbitrap FTMS.

Cited by 5 publications

References 52 publications

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Effective Amino Acid Sequencing of Intact Filgrastim by Multimodal Mass Spectrometry with Topdownr

Impact of Internal Fragments on Top-Down Analysis of Intact Proteins by 193 nm UVPD

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