Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation

Monoclonal antibodies (mAbs) are a rapidly advancing class of therapeutic glycoproteins that possess wide clinical utility owing to their biocompatibility, high antigen specificity, and targeted immune stimulation. These therapeutic properties depend greatly on the composition of the immunoglobulin G (IgG) structure, both in terms of primary sequence and post-translational modifications (PTMs); however, large-scale production in cell culture often results in heterogeneous mixtures that can profoundly affect clinical safety and efficacy. This places a high demand on analytical methods that afford comprehensive structural characterization of mAbs to ensure their stringent quality control. Here we report the use of targeted middle-down 193 nm ultraviolet photodissociation (UVPD) to provide detailed primary sequence analysis and PTM site localization of therapeutic monoclonal antibody subunits (∼25 kDa) generated upon digestion with recombinant immunoglobulin G-degrading enzyme of <i>Streptococcus pyogenes</i> (IdeS) followed by chemical reduction. Under optimal conditions, targeted UVPD resulted in approximately 60% overall coverage of the IgG sequence, in addition to unambiguous glycosylation site localization and extensive coverage of the antigen-binding complementarity determining regions (CDRs) in a single LC-MS/MS experiment. Combining UVPD and ETD data afforded even deeper sequencing and greater overall characterization of IgG subunits. Overall, this targeted UVPD approach represents a promising new strategy for the comprehensive characterization of antibody-based therapeutics

Selective 351 nm Photodissociation of Cysteine-Containing Peptides for Discrimination of Antigen-Binding Regions of IgG Fragments in Bottom-Up Liquid Chromatography–Tandem Mass Spectrometry Workflows

Despite tremendous inroads in the development of more sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC–MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine–peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of <i>b</i>- and <i>y</i>-type fragment ions, thus providing a facile means for differentiating cysteine–peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated

Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent

The labile nature of phosphoryl groups has presented a long-standing challenge for the characterization of protein phosphorylation via conventional mass spectrometry-based bottom-up proteomics methods. Collision-induced dissociation (CID) causes preferential cleavage of the phospho-ester bond of peptides, particularly under conditions of low proton mobility, and results in the suppression of sequence-informative fragmentation that often prohibits phosphosite determination. In the present study, the fragmentation patterns of phosphopeptides are improved through ion/ion-mediated peptide derivatization with 4-formyl-1,3-benezenedisulfonic acid (FBDSA) anions using a dual spray reactor. This approach exploits the strong electrostatic interactions between the sulfonate moieties of FBDSA and basic sites to facilitate gas-phase bioconjugation and to reduce charge sequestration and increase the yield of phosphate-retaining sequence ions upon CID. Moreover, comparative CID fragmentation analysis between unmodified phosphopeptides and those modified online with FBDSA or in solution via carbamylation and 4-sulfophenyl isothiocyanate (SPITC) provided evidence for sulfonate interference with charge-directed mechanisms that result in preferential phosphate elimination. Our results indicate the prominence of charge-directed neighboring group participation reactions involved in phosphate neutral loss, and the implementation of ion/ion reactions in a dual spray reactor setup provides a means to disrupt the interactions by competing hydrogen-bonding interactions between sulfonate groups and the side chains of basic residues

High-Throughput Bioconjugation for Enhanced 193 nm Photodissociation via Droplet Phase Initiated Ion/Ion Chemistry Using a Front-End Dual Spray Reactor

Fast online chemical derivatization of peptides with an aromatic label for enhanced 193 nm ultraviolet photodissociation (UVPD) is demonstrated using a dual electrospray reactor implemented on the front-end of a linear ion trap (LIT) mass spectrometer. The reactor facilitates the intersection of protonated peptides with a second population of chromogenic 4-formyl-1,3-benzenedisulfonic acid (FBDSA) anions to promote real-time formation of ion/ion complexes at atmospheric pressure. Subsequent collisional activation of the ion/ion intermediate results in Schiff base formation generated via reaction between a primary amine in the peptide cation and the aldehyde moiety of the FBDSA anion. Utilizing 193 nm UVPD as the subsequent activation step in the MS³ workflow results in acquisition of greater primary sequence information relative to conventional collision induced dissociation (CID). Furthermore, Schiff-base-modified peptides exhibit on average a 20% increase in UVPD efficiency compared to their unmodified counterparts. Due to the efficiency of covalent labeling achieved with the dual spray reactor, we demonstrate that this strategy can be integrated into a high-throughput LC-MS^<i>n</i> workflow for rapid derivatization of peptide mixtures

The GCN2-ATF4 Signaling Pathway Induces 4E-BP to Bias Translation and Boost Antimicrobial Peptide Synthesis in Response to Bacterial Infection

Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. Here, we use a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). We found that 4E-BP is induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5′ UTRs of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5′ UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis

Middle-Down 193-nm Ultraviolet Photodissociation for Unambiguous Antibody Identification and its Implications for Immunoproteomic Analysis

Mass spectrometry (MS) has emerged as a powerful tool within the growing field of immunoproteomics, which aims to understand antibody-mediated immunity at the molecular-level based on the direct determination of serological antibody repertoire. To date, these methods have relied on the use of high-resolution bottom-up proteomic strategies that require effective sampling and characterization of low abundance peptides derived from the antigen-binding domains of polyclonal antibody mixtures. Herein, we describe a method that uses restricted Lys-C enzymatic digestion to increase the average mass of proteolytic IgG peptides (≥4.5 kDa) and produce peptides which uniquely derive from single antibody species. This enhances the capacity to discriminate between very similar antibodies present within polyclonal mixtures. Furthermore, our use of 193-nm ultraviolet photodissociation (UVPD) improves spectral coverage of the antibody sequence relative to conventional collision- and electron-based fragmentation methods. We apply these methods to both a monoclonal and an antibody mixture. By identifying from a database search of approximately 15 000 antibody sequences those which compose the mixture, we demonstrate the analytical potential of middle-down UVPD for MS-based serological repertoire analysis

Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S–Se Bond Cleavage with 266 nm Ultraviolet Photodissociation

The tremendous number of peptides identified in current bottom-up mass spectrometric workflows, although impressive for high-throughput proteomics, results in little selectivity for more targeted applications. We describe a strategy for cysteine-selective proteomics based on a tagging method that installs a S–Se bond in peptides that is cleavable upon 266 nm ultraviolet photodissociation (UVPD). The alkylating reagent, <i>N</i>-(phenylseleno)­phthalimide (NPSP), reacts with free thiols in cysteine residues and attaches a chromogenic benzeneselenol (SePh) group. Upon irradiation of tagged peptides with 266 nm photons, the S–Se bond is selectively cleaved, releasing a benzeneselenol moiety corresponding to a neutral loss of 156 Da per cysteine. Herein we demonstrate a new MS/MS scan mode, UVPDnLossCID, which facilitates selective screening of cysteine-containing peptides. A “prescreening” event occurs by activation of the top N peptide ions by 266 nm UVPD. Peptides exhibiting a neutral loss corresponding to one or more SePh groups are reactivated and sequenced by CID. Because of the low frequency of cysteine in the proteome, unique cysteine-containing peptides may serve as surrogates for entire proteins. UVPDnLossCID does not generate as many peptide spectrum matches (PSMs) as conventional bottom-up methods; however, UVPDnLossCID provides far greater selectivity

UVnovo: A <i>de Novo</i> Sequencing Algorithm Using Single Series of Fragment Ions via Chromophore Tagging and 351 nm Ultraviolet Photodissociation Mass Spectrometry

<i>De novo</i> peptide sequencing by mass spectrometry represents an important strategy for characterizing novel peptides and proteins, in which a peptide’s amino acid sequence is inferred directly from the precursor peptide mass and tandem mass spectrum (MS/MS or MS³) fragment ions, without comparison to a reference proteome. This method is ideal for organisms or samples lacking a complete or well-annotated reference sequence set. One of the major barriers to <i>de novo</i> spectral interpretation arises from confusion of N- and C-terminal ion series due to the symmetry between <i>b</i> and <i>y</i> ion pairs created by collisional activation methods (or <i>c</i>, <i>z</i> ions for electron-based activation methods). This is known as the “antisymmetric path problem” and leads to inverted amino acid subsequences within a <i>de novo</i> reconstruction. Here, we combine several key strategies for <i>de novo</i> peptide sequencing into a single high-throughput pipeline: high-efficiency carbamylation blocks lysine side chains, and subsequent tryptic digestion and N-terminal peptide derivatization with the ultraviolet chromophore AMCA yield peptides susceptible to 351 nm ultraviolet photodissociation (UVPD). UVPD-MS/MS of the AMCA-modified peptides then predominantly produces <i>y</i> ions in the MS/MS spectra, specifically addressing the antisymmetric path problem. Finally, the program UVnovo applies a random forest algorithm to automatically learn from and then interpret UVPD mass spectra, passing results to a hidden Markov model for <i>de novo</i> sequence prediction and scoring. We show this combined strategy provides high-performance <i>de novo</i> peptide sequencing, enabling the <i>de novo</i> sequencing of thousands of peptides from an <i>Escherichia coli</i> lysate at high confidence