Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the tenth type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human EGFR, we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFRAddnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitope from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.
A fast, simple, sensitive, and low-cost method for electrochemical multianalyte immunoassay was developed by combining newly designed electric field-driven incubation with a screen-printed reagentless immunosensor array. The disposable array was prepared by immobilizing respectively horseradish peroxidase (HRP)-labeled antibodies modified gold nanoparticles in biopolymer/sol-gel modified electrodes to obtain direct electrochemical responses of HRP. Upon the formation of immunocomplexes, the responses decreased due to increasing spatial blocking and impedance. At a driving potential of 0.5 V, the incubation process could be accomplished within 2 min. Under optimal conditions, this method could simultaneously detect carbohydrate antigens 153, 125, and 199 and carcinoembryonic antigens ranging from 0.084 to 16, 0.11 to 13, and 0.16 to 15 U mL(-1) and 0.16 to 9.2 ng mL(-1) with a detection time of less than 5 min, and the detection limits corresponding to the signals of 3SD were 0.06, 0.03, and 0.10 U mL(-1) and 0.04 ng mL(-1), respectively. The disposable immunosensor array and simple detection system for fast measurement of panels of tumor markers show significant clinical value for application in cancer screening and provide great potential for convenient point-of-care testing and commercial application.
In
therapeutic monoclonal antibody (mAb) development, charge heterogeneity
of a mAb molecule is often associated with critical quality attributes
and is therefore monitored throughout development and during QC release
to ensure product and process consistency. Elucidating the cause of
each charge variant species is an involved process that often requires
offline fractionation by ion exchange chromatography (IEX) followed
by mass spectrometry (MS) analysis, largely due to the incompatibility
of conventional IEX buffers for direct MS detection. In this study,
we have developed a method that combines a generic strong cation exchange
(SCX) chromatography step with ultrasensitive online native MS analysis
(SCX-MS) optimized for mAb separation and detection. As demonstrated
by analyzing mAb molecules with a wide range of pI (isoelectric point)
values, the developed method can consistently achieve both high-resolution
IEX separation and ultrasensitive MS detection of low-abundance charge
variant species. Using this method, we analyzed the charge heterogeneity
of NISTmAb reference material 8671 (NISTmAb) at both whole antibody
and subdomain levels. In particular, due to the high sensitivity,
a nonconsensus Fab glycosylation site, present at a very low level
(<0.1%), was directly detected in the NISTmAb sample without any
enrichment. The structure and location of this Fab glycosylation was
further characterized by peptide mapping analysis. Despite the extensive
characterization of NISTmAb material in previous studies, this is
the first time that this Fab-glycosylated variant has been identified
in the NISTmAb, demonstrating the value of this new method in achieving
a more comprehensive characterization of charge heterogeneity for
therapeutic mAbs.
SUMMARY
In social interactions among mammals, individuals are recognized by olfactory cues, but identifying the key signals among thousands of compounds remains a major challenge. To address this need, we developed a new technique, Component-Activity Matching (CAM), to select candidate ligands that “explain” patterns of bioactivity across diverse complex mixtures. Using mouse urine from eight different sexes and strains, we identified 23 components to explain firing rates in seven of eight functional classes of vomeronasal sensory neurons. Focusing on a class of neurons selective for females, we identified a novel family of vomeronasal ligands, steroid carboxylic acids. These ligands accounted for much of the neuronal activity of urine from some female strains, were necessary for normal levels of male investigatory behavior of female scents and sufficed to trigger mounting behavior. CAM represents the first step towards an exhaustive characterization of the molecular cues for natural behavior in a mammalian olfactory system.
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