Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody

Zhang, Wei; Marzilli, Lisa A.; Rouse, Jason C.; Czupryn, Marta

doi:10.1016/s0003-2697(02)00394-9

Cited by 85 publications

(73 citation statements)

References 29 publications

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“…12,24,64,65 The LC-MALDI-TOF/TOF approach described here uses the Bruker DisulfideDetect software to automatically output semi-quantitative maps of the singly disulfide-bonded peptides within a single analysis. This method takes advantage of the partial reductive fragmentation of peptides at the disulfide bond by in-source decay in the MALDI source.…”

Section: Discussionmentioning

confidence: 99%

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

Resemann

Liu-Shin

Tremintin

et al. 2018

mAbs

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Human antibodies of the IgG2 subclass exhibit complex inter-chain disulfide bonding patterns that result in three structures, namely A, A/B, and B. In therapeutic applications, the distribution of disulfide isoforms is a critical product quality attribute because each configuration affects higher order structure, stability, isoelectric point, and antigen binding. The current standard for quantification of IgG2 disulfide isoform distribution is based on chromatographic or electrophoretic techniques that require additional characterization using mass spectrometry (MS)-based methods to confirm disulfide linkages. Detailed characterization of the IgG2 disulfide linkages often involve MS/MS approaches that include electrospray ionization or electron-transfer dissociation, and method optimization is often cumbersome due to the large size and heterogeneity of the disulfide-bonded peptides. As reported here, we developed a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. We established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

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Section: Discussionmentioning

confidence: 99%

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

Resemann

Liu-Shin

Tremintin

et al. 2018

mAbs

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“…As shown in Figure 1A, the signals that are present in one run, but not in the other, are considered to be disulfide bond related and can be collected for subsequent identification. RP-HPLC/MS was used to analyze the native and reduced Lys-C digest from an IgG4 monoclonal antibody (mAb) (Zhang et al 2002). By comparing the HPLC-UV chromatograms, the disulfidelinked peptides were localized, collected, and sequenced by offline nanoLC-MS/MS or Edman degradation.…”

Section: Profile Comparisonmentioning

confidence: 99%

Mass spectrometry-based strategies for protein disulfide bond identification

Tsai

Chen²,

Huang

2013

Reviews in Analytical Chemistry

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Abstract:The formation of disulfide bonds is critical for stabilizing protein structures and maintaining protein functions. It is important to understand the linkages between multiple cysteine residues within a protein. In this review, the analytical approaches using mass spectrometry (MS) for disulfide linkage assignment are classified and discussed. Enzymatic digestion under appropriate conditions followed by various MS detection strategies remains the primary method for cysteine linkage analysis. In-source decay (ISD) and electron transfer dissociation (ETD) have been used to generate significant peptide signals that indicate the identities of peptides involved in disulfide bonds. In addition, chemical labeling and software algorithms were also developed to facilitate the automation of disulfide bond analysis. For proteins with complex disulfide structure, methods involving partial reduction coupled with differential alkylation were demonstrated to be useful. In the past two decades, MS has become one of the most valuable tools for protein disulfide bond analysis. It provides irreplaceable information including the peptide backbone sequences as well as the cysteine connection pattern when coupling with appropriate sample preparations. The related approaches with their unique features can be applied for different aims such as structural characterization or functional studies of proteins.

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“…2(A)], the most notable difference being the lower abundance or the complete disappearance (depending on time of incubation) of the major cystine clusters and the appearance of two new species, m/z ¼ 1318. Table I Tables I and II. sequenced as residues [31][32][33][34][35][36][37][38][39][40][41][42][43] and [79-95], respectively, each containing a single intramolecular disulfide bridge (Table II). The native and recombinant SVN were indistinguishable with respect to the emergence of these new tryptic peptide species.…”

Section: Disulfide Interchange Within Cystine Clusters At Alkaline Phmentioning

confidence: 99%

“…Namely, a peak coeluting with PTH-Tyr (RT: $13.3 min), a unique peak eluting at RT: $14.2 min characteristic of the presence of cystine, and traces of PTH-anhydroSer (RT: $12.30 min) and PTH-Ser (RT: $7.30 min) that most likely result from beta-elimination/rehydration of di-PTH-Cystine during Edman degradation. [29][30][31] To increase the UV (k ¼ 269 nm) signal yield of di-PTH-Cystine, the sequencer reagent, R4A, 25% TFA/water, that contains 0.01% DTT as supplied by the manufacturer, was replaced by 25%, v/v, HPLCgrade TFA/water to eliminate DTT from the system. 30 Alkaline stability of disulfide-linked peptides SVN tryptic peptides of m/z ¼ 2511.0, m/z ¼ 2529.0, m/z ¼ 2719.1, and m/z ¼ 2737.1, isolated from pH 6 tryptic digests, were incubated at 37 C at pH 6 or pH 8 for various periods of time.…”

Section: Edman Degradationmentioning

confidence: 99%

Topology of the disulfide bonds in the antiviral lectin scytovirin

et al. 2010

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The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the ''cystine clusters'' [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/ Disclaimer: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.Abbreviations: Acm, acetamidomethyl; ATZ, 2-anilino-5-thiazolinone; AUFS, absorbance unit full scale; Boc-Gly-OCH 2 -PAM, t-butyloxycarbonyl-glycyl-4-(hydroxymethyl)phenylacetamidomethyl-copoly(styrene/1% divinylbenzene) resin; DIEA, diisopropylethylamine; ESI-Q-TOF, electrospray ionization quadrupole-time-of-flight mass spectrometry; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino) methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide; HFBA, heptafluorobutyric acid; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; pE, pyroglutamyl; PTH, phenylthiohydantoin; RP-HPLC, reversed-phase high pressure liquid chromatography; rSD1, recombinant/oxidatively folded N-terminal domain of SVN (residues 1-48Cys7Ser); R.T., retention time on HPLC; SD1A, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys26, Cys32-Cys38)]; SD1B, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys32, Cys26-Cys38)]; TCEP, tris(2-Carboxyethyl)phosphine; TFA, trifluoroacetic acid. Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584. Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require consider...

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Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody

Cited by 85 publications

References 29 publications

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

Mass spectrometry-based strategies for protein disulfide bond identification

Topology of the disulfide bonds in the antiviral lectin scytovirin

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