Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl₂ at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys¹⁴ to Cys⁷⁹, Cys⁵⁷ to Cys¹⁰⁸, and Cys⁶⁷ to Cys¹¹⁰. This disulfide structure is homologous to the NGF family of neurotrophic factors.     

Variant antibody identification by peptide mapping

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A‐purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H‐chain) band with a molecular weight slightly lower than that of the principal H‐chain band on SDS‐PAGE. The N‐terminal amino acid sequence of this minor H‐chain species indicated that at least the first 30 amino acids were identical to those of the antibody light‐chain (L‐chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg¹⁰⁸ of the L chain and Ala¹²⁴ of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful “first‐line” analytical tool in the characterization of the quality of the monoclonal antibody. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 485–488, 1999.     

Comparative peptide mapping of baculovirus polyhedrins

Amino terminals and two-dimensional high-voltage peptide maps of tryptic digests of polyhedrins from Heliothis armigera nuclear polyhedrosis virus (NPV), Heliothis zea NPV, and Anticarsa gemmatalis NPV were compared with previously characterized granulins and polyhedrins. Similarities and differences were detected in the tryptic maps, while each protein produced a unique peptide map composite. Amino-terminal determination of eight polyhedrins and granulins resulted in three different end groups.     

Purification,characterization and application of a novel antimicrobial peptide from Andrias davidianus blood

The Andrias davidianus has been known as a traditional Chinese medicine for a long time. Its blood is considered as a waste or by‐product of the meat production industry. Although there are reports on isolation of the antimicrobial peptides from different resources, there are no reports of their isolation from A. davidianus blood. In this work, an antimicrobial peptide, andricin B, was isolated from the blood of A. davidianus by an innovative method in which the magnetic liposome adsorption was combined with reversed‐phase high‐performance liquid chromatography. The structure, antimicrobial activity and safety of andricin B were further investigated. Amino acid sequence was determined by N‐terminal sequencing and found to be Gly‐Leu‐Thr‐Arg‐Leu‐Phe‐Ser‐Val‐Ile‐Lys. Circular dichroism (CD) spectra and prediction of three‐dimensional structure by bioinformatics software suggested the presence of a well‐defined random coil conformation. Andricin B was found to be active against all bacteria tested in this study as well as some fungi. The minimum inhibitory concentrations (MICs) were in the range 8–64 μg ml^?1. Moreover, the haemolytic testing also suggested that andricin B could be considered safe at the MICs. Finally, andricin B was shown to inhibit the growth of Staphylococcus aureus in the cooked meat of A. davidianus. This study shows that andricin B is a promising novel antimicrobial peptide that may provide further insights towards the development of new drugs.

Significance and Impact of the Study

This is the pioneer study on screening and isolation of antimicrobial peptide from the blood of Andrias davidianus. Here, we have developed a novel method by combining magnetic liposomes adsorption with reversed‐phase high‐performance liquid chromatography to purify and screen the antimicrobial peptides. From this screen, we identified a novel antimicrobial peptide which we name as andricin B. Andricin B is unique as it checks the growth of both Gram‐positive and Gram‐negative bacteria as well as few fungal species.     

Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at epsilon-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.     

MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies

Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step. The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads. This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.     

Characterization of a membrane-specific tubulin isoform by peptide mapping

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.     

Radioiodination and I-labeled peptide mapping of proteins on nitrocellulose membranes

A rapid procedure for generating dozens of ¹²⁵I-labeled peptide maps from a protein band excised from a single lane of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel has been developed. Proteins, which can be rapidly purified by 2× SDS-PAGE separation, are electroblotted onto nitrocellulose paper (NCP) and located by aqueous naphthol blue-black staining. All subsequent steps of radioiodination, and enzyme or chemical cleavage, are carried out on the NCP making it possible to test a variety of cleavage reagents on the same protein sample. The resultant peptidic residues, which can be separated by thin-layer electrophoresis-thin-layer chromatography (2D TLE-TLC), SDS-PAGE, or HPLC, can be used in comparative studies or they can be recovered for further structural and immunological analyses.     

Identification of the epitopes of calcitonin gene-related peptide (CGRP) for two anti-CGRP monoclonal antibodies by 2D NMR.

The interactions between calcitonin gene-related peptide and FAB fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (CB3 and CD1) have been studied at physiological pH using the ability of 1H NMR to detect selectively regions of dynamic flexibility. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C-terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle section of the sequence (22-31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.     

Nitrate reductase-deficient mutants in barley: enzyme stability and peptide mapping

Thermal stability and pH optima of NADH-nitrate reductase-associated cytochrome c reductase and FMNH₂-nitrate reductase from wild type, cv Steptoe or Winer, and mutants nar 1d, nar 1g, nar 1h, Xno 18 and Xno 19 were compared to determine if structural differences in the nitrate reductase protein could be detected. Also, the nitrate reductase-associated cytochrome c reductase from nar 1d was purified and compared with the wild type by peptide mapping. The pH optimum for FMNH₂-nitrate reductase from Steptoe and nar 1h, and for NADH-cytochrome c reductase from Steptoe, nar 1d, nar 1g and nar 2a was 7.5. Thermal stabilities of the nitrate reductase-associated activities (FMNH₂-nitrate reductase or NADH-cytochrome c reductase) from nar mutants were less than the Steptoe wild type, while Xno mutants were equal to the Winer wild type. Cleveland peptide maps of nar 1d NADH-cytochrome c reductase and Steptoe nitrate reductase were identicalwhen digested with endoprotease lys-C but were distinctly different in one peptide when digested with Staphylococcus aureus endoprotease V8. These results provide evidence that nar 1 gene codes for the nitrate reductase polypeptide.     

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy

Combination therapy is a fast-growing strategy to maximize therapeutic benefits to patients. Co-formulation of two or more therapeutic proteins has advantages over the administration of multiple medications, including reduced medication errors and convenience for patients. Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. We present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. We developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)–based method to monitor site-specific deamidation. Our approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.     

Development and characterization of a novel anti-IgE monoclonal antibody

IgE is the central macromolecular mediator responsible for the progression of allergic reactions. Omalizumab (Xolair) is a humanized monoclonal anti-IgE antibody directed at the FcεRI-binding domain of human IgE, which represents a novel therapeutic approach in the management of asthma. In this study, we developed a monoclonal antibody (7A5) against human IgE via hybridoma technique. Our data showed that 7A5 could inhibit free IgE molecules to bind to receptors without affecting IgE already bound to cellular receptors. Importantly, 7A5 was able to inhibit IgE-induced histamine release of basophilic leukemia cells. Next, the phage display peptide library technology was employed to select peptides binding to 7A5 and a striking peptide sequence motif was recovered, which is homologous to the sequence ³⁹¹KQR³⁹³ within the Cε3 domain of IgE-Fc, Our results further indicated that 7A5 specifically bound to the synthesized peptide “³⁸⁸KEEKQRN³⁹⁴” containing the ³⁹¹KQR³⁹³ motif in IgE-Fc. The epitope of 7A5 was found to be spatially close to the FcεRI-binding site, suggesting that 7A5 binding to IgE might block IgE binding to receptors via steric hindrance. The anti-IgE monoclonal antibody 7A5 may have the potential to be developed as a therapeutic agent for the treatment of allergic diseases.     

Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.

Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.     

Tightly bound nonprotamine proteins from ram sperm nuclei studied by one- and two-dimensional peptide mapping

The tightly bound proteins of ram sperm nuclei (TBSP) have been recovered as a fraction co-sedimenting with DNA after high salt-urea deprotamination of the nuclei. TBSP were studied by two-dimensional (2D)tryptic peptide mapping and by one-dimensional (1D)-partial proteolysis mapping. The 2D maps revealed a strong homology among the proteins, irrespective of substantial differences in their molecular masses. This homology was supported also by the 1D-mapping data.The 2D-tryptic maps of TBSP were compared to those of lamb liver lamins but no apparent similarity was detected. TBSP were found to react positively to a test for the presence of carbohydrate residues, suggesting that these proteins are glycoproteins as established earlier for the lamins.The 2D maps of several proteins of seminal plasma origin, used as a control, displayed completely different peptide profiles.     

Human CD30: structural implications from epitope mapping and modeling studies

The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.     

Enzymatic cleavage and HPLC peptide mapping of proteins

Detailed procedures are described for successfully digesting reasonably small quantities (i.e., usually >10 pmol) of proteins with a variety of proteases and for then isolating the resulting peptides by reversephase HPLC. Since sodium dodecyl sulfate-polyacrylamide, gel electrophoresis (SDS-PAGE) appears to be the current method of choice for final purification of proteins for structural analysis, special attention is given to carrying out in-gel proteolytic digests on SDS-PAGE-separated proteins that have usually been stained with Coomassie Blue. A compilation of data from nearly 200 “unknown” samples is used to help provide realistic expectations with respect to the results that are likely to be obtained from carrying out in-gel proteolytic digests on large numbers of proteins.     

Identification and mapping of a linear epitope of centromere protein F using monoclonal antibodies

Autoantibodies against centromere protein ‐F have been reported to be associated with various types of cancer with poor prognosis. The characterization of these autoantibody specificities is important in both diagnostics and basic research. In this study, we mapped the epitope (NELSRIRSEKA) of two monoclonal centromere protein F antibodies. The epitope was localized by screening of overlapping peptides followed by a fast and efficient estimation of the minimal peptide length required for antibody recognition, based on the screening of terminally truncated resin‐bound peptide analogs. The epitope was determined through competitive inhibition assays of systematically truncated free peptides. In addition, the importance of the involved amino acid side chains of the identified epitope was determined through competitive inhibition assays using alanine‐substituted analogs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Direct mass spectrometric characterization of disulfide linkages

Disulfide linkage is critical to protein folding and structural stability. The location of disulfide linkages for antibodies is routinely discovered by comparing the chromatograms of the reduced and non-reduced peptide mapping with location identification confirmed by collision-induced dissociation (CID) mass spectrometry (MS)/MS. However, CID product spectra of disulfide-linked peptides can be difficult to interpret, and provide limited information on the backbone region within the disulfide loop. Here, we applied an electron-transfer dissociation (ETD)/CID combined fragmentation method that identifies the disulfide linkage without intensive LC comparison, and yet maps the disulfide location accurately. The native protein samples were digested using trypsin for proteolysis. The method uses RapiGest SF Surfactant and obviates the need for reduction/alkylation and extensive sample manipulation. An aliquot of the digest was loaded onto a C₄ analytical column. Peptides were gradient-eluted and analyzed using a Thermo Scientific LTQ Orbitrap Elite mass spectrometer for the ETD-triggered CID MS³ Wypych J, Li M, Guo A, Zhang Z, Martinez T, Allen MJ, Fodor S, Kelner DN, Flynn GC, Liu YD, et al. Human IgG2 antibodies display disulfide-mediated structural isoforms. J Biol Chem. 2008;283:16194–205. doi:10.1074/jbc.M709987200. PMID:18339624[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] experiment. Survey MS scans were followed by data-dependent scans consisting of ETD MS² scans on the most intense ion in the survey scan, followed by 5 MS³ CID scans on the 5 most intense ions in the ETD MS² scan. We were able to identify the disulfide-mediated structural variants A and A/B forms and their corresponding disulfide linkages in an immunoglobulin G2 monoclonal antibody with λ light chain (IgG2λ), where the location of cysteine linkages were unambiguously determined.