Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins

Wei, Benqian; Zenaidee, Muhammad A.; Lantz, Carter; Williams, Brad J.; Totten, Sarah; Loo, Rachel R. Ogorzalek; Loo, Joseph A.

doi:10.1039/d2an01517j

Cited by 14 publications

(20 citation statements)

References 70 publications

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“…For terminal fragments to cover the disulfide bonded sequence region, cleavages of both protein backbone and disulfide bonds are required. However, internal fragments can access these highly constrained regions without the need of breaking disulfide bonds, thus having the potential to substantially enhance sequence information on intact mAbs . Additionally, internal fragments contain two cleavage sites while terminal fragments only contain one, making internal fragments naturally more information-rich than terminal fragments.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, our group demonstrated that internal fragments that retain intact disulfide bonds can be used to determine S−S connectivity of intact proteins (Figure S3). 67 This encourages us to explore the utility of such fragments to determine intrachain S−S connectivity of intact NIST mAb, which is comprised of 16 disulfide bonds. HCD is known to only cleave the protein backbone while maintaining the integrity of disulfide bonds; therefore, we applied HCD on the intact NIST mAb to generate such fragments to determine S−S connectivity.…”

Section: Internal Fragments Can Identify Mab Ptms Including Intrachai...mentioning

confidence: 99%

“…Previous studies have shown that including internal fragment assignments in TD-MS can enhance the sequence information obtained from intact proteins, − from protein complexes, , and on a proteome-wide scale . Specifically, internal fragments have been demonstrated to aid the TD-MS characterization of disulfide-intact proteins, ,, inspiring us to investigate the application of internal fragments in TD-MS of mAbs and ADCs, which contain a large number of disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

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Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody–Drug Conjugates

et al. 2023

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Monoclonal antibodies (mAbs) and antibody−drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-ofprinciple study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules.

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Section: Resultsmentioning

confidence: 99%

Section: Internal Fragments Can Identify Mab Ptms Including Intrachai...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody–Drug Conjugates

et al. 2023

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“…Furthermore, the data appeared to plateau for the HCD and CID comparisons of ACN/H 2 O, suggesting that additional time may not be sufficient to overcome barriers between structural states. To provide additional energy toward annealing, we increased collisional activation beyond the threshold for fragmentation of the protein, while retaining the 100 ms activation time, and then reisolated the unfragmented population to be fragmented in MS 3 . While this does result in some decrease in signal, additional heating of the ions should be attained.…”

Section: Journal Of the Americanmentioning

confidence: 99%

“…Further information can be obtained by comparison of the N+1 and N+2 forms, which yield effect sizes below 0.55 for both myoglobin and β-hemo. The N charge state is isolated in MS 2 without PTCR (red) and is fragmented in MS 3 . The N+1 and N+2 charge states are isolated in MS 2 and undergo PTCR reactions to create reduced charge state distributions (yellow and blue).…”

Section: Journal Of the Americanmentioning

confidence: 99%

Fragment Ion Abundance Reveals Information about Structure and Charge Localization in Highly Charged Proteins

Shoff

Julian

2023

J. Am. Soc. Mass Spectrom.

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Top-down mass spectrometry (MS) is a versatile tool that has been employed to investigate both protein sequence and structure. Although a variety of different fragmentation methods are available in top-down MS that can potentially yield structural information, quantifying differences between spectra remains challenging. Herein, we show that subtle differences in spectra produced by a variety of fragmentation methods are surprisingly sensitive to protein structure and/or charge localization, even in highly unfolded proteins observed in high charge states. In addition to exposing information about the protein structure, differences in fragmentation also reveal insight into the mechanisms underlying the dissociation methods themselves. The results further reveal that small changes in experimental parameters (such as the addition of methanol instead of acetonitrile) lead to changes in structure that are reflected in statistically reproducible differences in dissociation. Collisional annealing of structurally dissimilar ions in the gas phase eventually leads to dissociation spectra that are indistinguishable, suggesting that structural differences can be erased by sufficient thermal activation. Additional experiments illustrate that identical charge states of the same protein can be distinguished if those produced directly by electrospray are compared to ions manipulated by in vacuo proton-transfer charge reduction. Overall, the results show that subtle differences in both three-dimensional structure and charge-site localization can influence the abundance of fragment ions produced by top-down MS, including dissociation methods not typically thought to be structurally sensitive.

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Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows

Beaumal,

Deslignière,

Diemer

et al. 2023

Anal Bioanal Chem

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Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins

Abstract: Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass...

Cited by 14 publications

References 70 publications

Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody–Drug Conjugates

Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody–Drug Conjugates

Fragment Ion Abundance Reveals Information about Structure and Charge Localization in Highly Charged Proteins

Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows

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