Identification and Sequencing of N-Terminal Peptides in Proteins by LC-Fluorescence-MS/MS: An Approach to Replacement of the Edman Degradation.

Automated Edman degradation for sequencing is the classic tool for determining the N-terminal sequences of proteins. Although it has been complemented or largely replaced by a variety of mass spectrometric methods in recent years, there is no simple mass spectrometric method for identification and de novo determination of the N-terminal sequence of a protein. Here, we present a simple and reliable approach which identifies N-terminal peptides of proteins by selectively labeling the N-terminal α-amino groups with a fluorescent reagent followed by enzyme digestion and tandem mass spectrometric analysis using an LC-MS/MS system coupled with a fluorescence detector. This approach also includes a differential peptide mapping step for identification of pyroglutamate-blocked N-terminal peptides. In addition, the strategy can be applied to proteins in gel. With this approach, complete N-terminal sequences of proteins can be obtained precisely and efficiently.

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies.

Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains - two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cε2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).

Determination of the Disulfide Structure of Murine Meteorin, a Neurotrophic Factor, by LC-MS and Electron Transfer Dissociation-High-Energy Collisional Dissociation Analysis of Proteolytic Fragments.

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.

Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis Using an Orbitrap Fusion Mass Spectrometer.

Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their molecular masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, especially in CDRs (complementarity determining regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, we describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissociation) multistage mass spectrometric analysis (HCD-MSn) and ETD (electron transfer dissociation)-HCD MS3 analysis using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides. The merits and limitations of this Leu/Ile discrimination approach are evaluated. Using the new approach, along with proposed decision-making guidelines we unambiguously identified every Leu/Ile residue in peptides containing up to five Leu/Ile residues and molecular masses up to 3000 Da. In addition, we have demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly determined using this approach. Our results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry. In principle, this integrated, online LC-MS approach for Leu/Ile assignment can be applied to de novo sequencing of any protein or peptide.

Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells.

Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA(Asn) rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.