De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand

Pham, Victoria C.; Henzel, William J.; Arnott, David; Hymowitz, S.G.; Sandoval, Wendy; Truong, Bao-Tran H.; Lowman, Henry B.; Lill, Jennie R.

doi:10.1016/j.ab.2006.02.001

Cited by 30 publications

(29 citation statements)

References 37 publications

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“…In the case of the 6E10 heavy chain, only the variable region V H (1-50) spanning the CDR1 (residues 26-35) could be assigned by searching the MS/MS data against the NCBInr database. The fragment ion spectra (Supporting Information) for peptides containing the regions V H (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), V H (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) and V H (39-50) are consistent with the sequence indicated in Figure 1. According to the Kabat rules, the V H CDR1, with a typical length of 10-12 amino acids, is located four residues after the first cysteine of the variable region (CXXX) and is followed always by a tryptophane (20,21).…”

Section: Primary Structure Determination Of the 6e10 Heavy Chainmentioning

confidence: 61%

“…In these studies, they used a combination of proteolytic and chemical digestions, Edman degradation, and mass spectrometry (26). However, a substantial portion of the data was derived from Edman sequence analysis, while mass spectrometry was rather inefficient in providing unambiguous structural information about the antigen binding regions.…”

Section: Introductionmentioning

confidence: 99%

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Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

et al. 2008

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Using the "bottom-up" approach and liquid chromatography in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-β-amyloid (1-17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high performance LC-tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10-antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyro-glutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig V L genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core fucosylated, biantennary, complex type structures containing zero to two galactose residues.

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Section: Primary Structure Determination Of the 6e10 Heavy Chainmentioning

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

et al. 2008

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“…In these cases, informed assignment may be guided by the sequence of a candidate germline gene segment, but when these sequence ambiguities map to hypervariable complementarity-determining regions (CDRs) of the antibody, this is not necessarily possible. A number of reports have described methods for partial de novo sequencing of antibody V-domains, [1][2][3][4][5][6] but obtaining the complete sequences necessary to enable reverse engineering of a mAb from protein material remains a very challenging undertaking.…”

Section: Introductionmentioning

confidence: 99%

“…However, this methodology was unable to determine the sequence of the CDR-H3 region, for which they had to resort to unspecified techniques, which may have included the use of non-MS based methods. 5 In the case of Sousa et al, 6 chemical modification of Cys residues to thialysine, which can by cleaved by Lys-specific proteases, was used to help with identification of peptides containing regions of CDR1 and/or CDR3, and isotopic labeling of Lys and Arg residues in a tentative draft construct was used to identify regions of sequence errors by comparing digests of this material to the parental antibody by LC/MS/MS. As these studies showed, MSbased de novo sequencing can readily provide 90-98% of the V-domain sequences, but elucidating the residual sequence, which is typically within critical CDRs, is challenging and would benefit from complementary techniques that can quickly survey a large number of potential sequence solutions.…”

Section: Introductionmentioning

confidence: 99%

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

Rickert¹,

Grinberg²,

Woods³

et al. 2016

mAbs

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(2016) Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies, mAbs, 8:3, 501-512, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTThe enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

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“…De novo sequencing finds use in special applications for which protein databases are difficult to obtain. These applications include unsequenced organisms [31], biotech products such as monoclonal antibodies [3,28], phosphopeptide epitopes [13], endogenous antibodies [29], and peptide toxins [2,27,37].…”

Section: Introductionmentioning

confidence: 99%

Constrained De Novo Sequencing of Peptides with Application to Conotoxins

Bhatia

Kil

Ueberheide

et al. 2011

Lecture Notes in Computer Science

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Abstract. We describe algorithms for incorporating prior sequence knowledge into the candidate generation stage of de novo peptide sequencing by tandem mass spectrometry. We focus on two types of prior knowledge: homology to known sequences encoded by a regular expression or position-specific score matrix, and amino acid content encoded by a multiset of required residues. We show an application to de novo sequencing of cone snail toxins, which are molecules of special interest as pharmaceutical leads and as probes to study ion channels. Cone snail toxins usually contain 2, 4, 6, or 8 cysteine residues, and the number of residues can be determined by a relatively simple mass spectrometry experiment. We show here that the prior knowledge of the number of cysteines in a precursor ion is highly advantageous for de novo sequencing.

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De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand

Cited by 30 publications

References 37 publications

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

Constrained De Novo Sequencing of Peptides with Application to Conotoxins

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