Protein derivatization and sequential ion/ion reactions to enhance sequence coverage produced by electron transfer dissociation mass spectrometry

Anderson, Lissa C.; English, Ann M.; Wang, Wei Han; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.

doi:10.1016/j.ijms.2014.06.023

Cited by 37 publications

(40 citation statements)

References 16 publications

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“…1C is an average of 20 spectra like that shown in Fig 1A and represents a cumulative acquisition time of 11.2 s (0.56 s/spectrum). The use and benefits of IIPT to charge reduce ETD-produced fragment ions have been described previously (1,23). Briefly, following fragmentation by ETD, fragment ions are allowed to react with a proton transfer reagent.…”

Section: Multiple Fills and Iipt-a Thermo Fisher Scientific Orbitrapmentioning

confidence: 99%

“…Histones comprise the principal protein component of chromatin and are involved in the regulation of gene expression (3,4). This epigenetic regulation is achieved through complex patterns of post-translational modifications (PTMs), 1 the incorporation of histone variants, and through controlled histone proteolysis (5)(6)(7)(8)(9)(10). Comprehensive characterization of histones by mass spectrometry (MS) has proven technically difficult for a number of reasons.…”

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confidence: 99%

“…We have previously described modifications to a Thermo Fisher Scientific Orbitrap Velos Pro TM mass spectrometer that make it possible to perform multiple fills of the C-trap with product ions from sequential ion/ion reactions of intact proteins prior to high resolution mass analysis (1,21). The result is a dramatic enhancement of the observed fragment ion current and significant improvement of signal-to-noise ratios (SNRs) in MS/MS spectra.…”

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confidence: 99%

“…The front-end ETD (FETD) source can also be used to ionize ion/ion proton transfer (IIPT) reagents. IIPT reactions are used to disperse fragment ions over the available m/z range in a controlled manner and to simplify ETD fragment ion spectra (1,22,23). Additionally, we have enabled parallel ion parking, first reported by McLuckey and co-workers (24,25), which involves harmonic excitation of selected ions within the ion trap to reduce their reactivity in gas-phase ion/ion reactions.…”

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confidence: 99%

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Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Anderson

Karch

Ugrin

et al. 2016

Molecular & Cellular Proteomics

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Chromatin is the structural framework that packages DNA into chromosomes within the nucleus of a cell (2). Histones comprise the principal protein component of chromatin and are involved in the regulation of gene expression (3,4). This epigenetic regulation is achieved through complex patterns of post-translational modifications (PTMs), 1 the incorporation of histone variants, and through controlled histone proteolysis (5-10). Comprehensive characterization of histones by mass spectrometry (MS) has proven technically difficult for a number of reasons. Traditional methods (bottom-up MS) of sequence determination and PTM site localization are not practical. Histone N-terminal regions are rich in lysine and arginine residues, and thus proteolysis using trypsin generates peptides that are too small or that are poorly retained on reversephase HPLC C18 resins for subsequent MS detection (11). With the advent of electron transfer dissociation (ETD) and more efficient electron capture dissociation fragmentation methods, which are better suited for larger, more highly charged peptides (12, 13), several studies utilizing other endoproteases to generate longer peptides have emerged (14 -16). Although these methodologies do well to preserve the combinatorial PTM profiles of histone tails, in some cases it is still impossible to identify the proteoforms from which these peptides originate. This is why analyzing histones intact, as they exist in the cells from which they are derived, is the best method for identifying unique histone proteoforms.The results of several recent studies involving top-down analyses of histones highlight the complexity of the histone From the ‡Department

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Section: Multiple Fills and Iipt-a Thermo Fisher Scientific Orbitrapmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Anderson

Karch

Ugrin

et al. 2016

Molecular & Cellular Proteomics

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“…We then targeted these unique peptides (nonreduced form) for sequence identification. A combination of ETD and gas phase IIPT reactions on these target ions allowed us to read amino acid sequences from the two N termini and two C termini of the disulfide-bonded peptides (22).…”

Section: Resultsmentioning

confidence: 99%

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Zhang

English

Bai

et al. 2016

Molecular & Cellular Proteomics

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Methodology for sequence analysis of ϳ150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 M urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3-9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous posttranslational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of Nlinked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion-ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides. Molecular & Cellular Proteomics

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Site‐Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation

et al. 2015

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Many proteins suffer from sub-optimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom transfer radical polymerization (ATRP) at the N-terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by reaction of the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methylacrylate) with a 1 carbon spacer (C1) (PCBMA), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of 1 carbon (C1) and 2 carbon (C2) spacer. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar MW. The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

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Protein derivatization and sequential ion/ion reactions to enhance sequence coverage produced by electron transfer dissociation mass spectrometry

Cited by 37 publications

References 16 publications

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Site‐Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation

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