Structural characterization by mass spectrometry of native and recombinant human relaxin

Stults, John T.; Bourell, James H.; Canova-Davis, Eleanor; Ling, Victor; Laramee, Gary R.; Winslow, John; Griffin, Patrick R.; Rinderknecht, Ernst; Vandlen, Richard

doi:10.1002/bms.1200191105

Cited by 67 publications

(35 citation statements)

References 39 publications

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“…Several authors have found that tandem mass spectrometry, employing highenergy collision-induced dissociation, is a useful method for the characterization of disulfide-bridged peptides. Stults et al (265) applied MSlMS for the characterization of tryptic peptides during FAB analysis of recombinant human relaxin, a peptide hormone that induces ripening of the cervix prior to childbirth. The three disulfide bonds were shown to match those of insulin.…”

Section: Disulfide Bondsmentioning

confidence: 99%

Mass spectrometry of sulfur‐containing compounds in organic and bioorganic fields

Bortolini¹,

Fogagnolo²

1995

Mass Spectrometry Reviews

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This survey covers the mass spectrometic properties of organosulfur compounds containing the sulfide, disulfide, and sulfone functional groups. Attention is paid to the uni‐ and biomolecular chemistry of both positive and negative ions, as well as to the reactions of ions, with sulfur containing species, in the gas phase. The role of other functional groups on the decomposition of organic S, SO, SO2 containing gaseous ions is also discussed. The selection of sulfur containing compounds includes many naturally occurring and pharmaceutically significant products, that will be presented in the following order: alkaloids, amino acids, peptides, proteins, carbohydrates, steroids, and metabolites. © 1996 John Wiley & Sons, Inc.

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Section: Disulfide Bondsmentioning

confidence: 99%

Mass spectrometry of sulfur‐containing compounds in organic and bioorganic fields

Bortolini¹,

Fogagnolo²

1995

Mass Spectrometry Reviews

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“…Previously, when magnetic sector instruments were widely used, high-energy CAD (HE CAD) was utilized to produce cleavage of numerous disulfide bonds in peptides. [8] The abundance of this fragmentation channel compared to lowenergy CAD has been explained by electronic excitation. Today, HE CAD can only be found in TOF/TOF instruments that are more suitable for peptides than for proteins, and for which MS n abilities are limited above n = 2.…”

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confidence: 99%

Facile Disulfide Bond Cleavage in Gaseous Peptide and Protein Cations by Ultraviolet Photodissociation at 157 nm

et al. 2005

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Disulfide bonds formed between cysteine residues stabilize the native structure of many proteins.[1] These covalent crosslinkages are part of the tertiary structure and introduce conformational constraints to the polypeptide architecture, thus improving the thermodynamic stability. Mass spectrometry has become increasingly important in the characterization and identification of proteins as a result of its capability for highly specific measurements on relatively small amounts of analyte. Protein characterization and identification relies on database searches using primary sequence information obtained by tandem mass spectrometry (MS n or MS/MS). The top-down approach [2] applied to intact proteins ions is an efficient MS/MS method which is potentially suited for the high-throughput characterization of proteins. The problems addressed by the top-down approach include verification of the protein sequence, localization of errors in DNA-predicted sequences, de novo sequencing, as well as the analysis of pre-or posttranslational modifications (PTMs). However, such PTMs as disulfide bonds still remain a challenge for traditional MS/MS techniques that utilize vibrational excitation (VE), as they break peptide bonds preferentially in protonated polypeptides and leave the disulfide bonds intact.[3] As a result, information on the primary sequence is limited to parts of the protein that is not constrained by disulfide bonds. This restriction complicates primary sequence elucidation and limits the utility of automated database searches. The problem of disulfide bonds is solved conventionally by their reduction and alkylation [4] prior to MS/MS measurements. However, the additional step involving solution-phase chemistry, including subsequent purification, greatly increases the analysis time and the risk of sample loss. Another approach is to use an alternative iondissociation strategy. Chrisman and McLuckey have shown that VE of deprotonated polypeptides induced by collisionally activated dissociation (CAD) [5] breaks disulfide bonds with high efficiency.[6] However, CAD of peptide anions often produces complicated mass spectra that, together with backbone cleavages, contain significant amounts of neutral mass losses and rearrangement products which make such MS/MS spectra of limited value for sequencing. Electron-capture dissociation (ECD) [7] stands alone in its ability to cleave SÀS bonds preferentially in peptide polycations (Scheme 1).[3] A drawback of this technique is that the capture of a low-energy electron by multiply charged polypeptides results in charge reduction prior to bond cleavage, which in turn leads to a charge decrease in one of the two fragments. As species need to contain at least one charge to be detectable in mass spectrometry, the charge reduction in ECD can lead to a loss of information. Previously, when magnetic sector instruments were widely used, high-energy CAD (HE CAD) was utilized to produce cleavage of numerous disulfide bonds in peptides.[8] The abundance of this fragmentation channel comp...

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“…Alignment of the human relaxin sequence with insulin reveals that the cysteine residues involved in the intermolecular linkages are in the same position (Nguyen and Shire 1996). A process was developed at Genentech to express rDNA-derived A and B chains in E. coli and then to recombine the individually purified chains into a biologically functional molecule (Stults et al 1990). Early characterization of human relaxin by size exclusion chromatography (SEC-HPLC) suggested that the molecule exists in the monomeric form (data not shown).…”

Section: Self-association Of Small Non-glycosylated Proteinsmentioning

confidence: 99%

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Yadav¹,

Li²,

Scherer³

et al. 2013

Biophys Rev

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Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibodyantigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

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Structural characterization by mass spectrometry of native and recombinant human relaxin

Cited by 67 publications

References 39 publications

Mass spectrometry of sulfur‐containing compounds in organic and bioorganic fields

Mass spectrometry of sulfur‐containing compounds in organic and bioorganic fields

Facile Disulfide Bond Cleavage in Gaseous Peptide and Protein Cations by Ultraviolet Photodissociation at 157 nm

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

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