Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

Comprehensive Middle-Down Mass Spectrometry Characterization of an Antibody-Drug Conjugate by Combined Ion Activation Methods.

Antibody-drug conjugates (ADCs) are an increasingly prevalent drug class utilized as chemotherapeutic agents. The complexity of ADCs, including their large size, array of drug conjugation sites, and heterogeneous compositions containing from zero to several payloads, demands the use of advanced analytical characterization methods. Tandem mass spectrometry (MS/MS) strategies, including a variety of bottom-up, middle-down, and even top-down approaches, frequently applied for the analysis of antibodies are increasingly being adapted for antibody-drug conjugates. Middle-down tandem mass spectrometry, often focusing on the analysis of ∼25 kDa protein subunits, offers the potential for complete sequence confirmation as well as the identification of multiple conjugation states. While middle-down studies have been extensively developed for monoclonal antibodies, middle-down characterization of ADCs has been limited by the high complexity of the drug molecules. This study seeks to bridge the gap by utilizing a combination of 193 nm ultraviolet photodissociation (UVPD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). The compilation of these MS/MS methods leads to high sequence coverages of 60-80% for each subunit of the ADC. Moreover, the combined fragmentation patterns provide sufficient information to allow confirmation of both the sequence of the complementarity-determining regions and the payload conjugation sites.

Molecular determinants of species-specific activation or blockade of TRPA1 channels.

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

Protein fragmentation via liquid chromatography-quadrupole time-of-flight mass spectrometry: the use of limited sequence information in structural characterization.

Fragmentation and "top-down" sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse "clean" solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top-down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC-MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top-down sequencing to problems in structural characterization, protein purification, and biomarker identification.