Middle Down Research Articles

Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows.

Antibody-drug conjugates (ADCs) are highly complex proteins mainly due to the structural microvariability of the mAb, along with the additional heterogeneity afforded by the bioconjugation process. Top-down (TD) and middle-down (MD) strategies allow the straightforward fragmentation of proteins to elucidate the conjugated amino acid residues. Nevertheless, these spectra are very crowded with multiple overlapping and unassigned ion fragments. Here we report on the use of dedicated software (ClipsMS) and application of proton transfer charge reduction (PTCR), to respectively expand the fragment ion search space to internal fragments and improve the separation of overlapping fragment ions for a more comprehensive characterization of a recently approved ADC, trastuzumab deruxtecan (T-DXd). Subunit fragmentation allowed between 70 and 90% of sequence coverageto be obtained. Upon addition of internal fragment assignment, the three subunits were fully sequenced, although internal fragments did not contribute significantly to the localization of the payloads. Finally, the use of PTCR after subunit fragmentation provided a moderate sequence coverage increase between 2 and 13%. The reaction efficiently decluttered the fragmentation spectra allowing increasing the number of fragment ions characteristic of the conjugation site by 1.5- to 2.5-fold. Altogether, these results show the interest in the implementation of internal fragment ion searches and more particularly the use of PTCR reactions to increase the number of signature ions to elucidate the conjugation sites and enhance the overall sequence coverage of ADCs, making this approach particularly appealing for its implementation in R&D laboratories.

Mass spectrometry characterization of antibodies at the intact and subunit levels: From targeted to large-scale analysis

Antibodies are one of the most formidable molecular weapons available to our immune system. Their high specificity against a target (antigen) and capability of triggering different immune responses (e.g., complement system activation and antibody-dependent cell-mediated cytotoxicity) make them ideal drugs to fight many different human diseases. Currently, both monoclonal antibodies and more complex molecules based on the antibody scaffold are used as biologics. Naturally, such highly heterogeneous molecules require dedicated analytical methodologies for their accurate characterization. Mass spectrometry (MS) can define the presence and relative abundance of multiple features of antibodies, including critical quality attributes. The combination of small and large variations within a single molecule can only be determined by analyzing intact antibodies or their large (25–100 kDa) subunits. Hence, top-down (TD) and middle-down (MD) MS approaches have gained popularity over the last decade. In this Young Scientist Feature we discuss the evolution of TD and MD MS analysis of antibodies, including the new frontiers that go beyond biopharma applications. We will show how this field is now moving from the “quality control” analysis of a known, single antibody to the high-throughput investigation of complex antibody repertoires isolated from clinical samples, where the ultimate goal is represented by the complete gas-phase sequencing of antibody molecules without the need of any a priori knowledge.

Mimicking LysC Proteolysis by 'Arginine Modification-cum-Trypsin Digestion': Comparison of Bottom-up & Middle-down Proteomic Approaches by ESI Q-TOF MS.

Middle-down (MD) proteomics is an emerging approach for reliable identification of post-translational modifications and isoforms, as this approach focuses on proteolytic peptides containing > 25-30 amino acid residues (a.a.r.), which are longer than typical tryptic peptides. Such longer peptides can be obtained by AspN, GluC, and LysC proteases. Additionally, some special proteases were developed specifically to effect MD approach, e.g., OmpT, Sap9, etc. However, these proteases are expensive. Herein we report a cost-effective strategy 'arginine modification- cum trypsin digestion', which can produce longer tryptic peptides resembling LysC peptides derived from proteins. The aim of this study is to obtain proteolytic peptides that resemble LysC peptides by using 'trypsin', which is a less expensive protease. This strategy is based on the simple principle that trypsin cannot act at the C-termini of those arginines in proteins, whose sidechain guanidine groups are modified by 1,2-cyclohexanedione or phenylglyoxal. As a proof of concept, we demonstrate this strategy on four models: β-casein (bovine), β- lactoglobulin (bovine), ovalbumin (chick) and transferrin (human), by electrospray ionization-mass spectrometry (ESI-MS) involving hybrid quadrupole time-of-flight. From the ESI-MS of these models, we obtained several arginine modified tryptic peptides, whose lengths are in the range of 30-60 a.a.r. The collision induced dissociation MS/MS characteristics of some of the arginine modified longer tryptic peptides are compared with the unmodified standard tryptic peptides. The strategy demonstrated in this proof-of-concept study is not only useful to obtain longer tryptic peptides that mimic LysC proteolytic peptides, but also facilitates in enhancing the probability of missed cleavages by the trypsin. Hence, this method aids in evading the possibility of obtaining very short peptides that are <5-10 a.a.r. Therefore, this is indeed a cost-effective alternative/ substitute for LysC proteolysis and, in turn, for those MD proteomic studies that utilize LysC. Additionally, this methodology can be fruitful for mass spectrometry-based de novo protein and peptide sequencing.

Beneficial role of batter pile in slope under infinite uniform surcharge loading

Batter pile is used to find a more effective way to stabilize the slope. Thus, these two questions “where is the optimal location of batter piles on earth slopes?” and “which inclination angle is most beneficial for pile in earth slopes?” arise. The numerical simulation was conducted to investigate the performance of slope reinforced by batter pile. The surcharge loading, inclination angle and pile location were varied while keeping geometry of the slope and the soil property constant. The surcharge loading was applied on the slope crest by steps. The inclination angle varied from 0° to 20°. The pile was placed at middle-down (MD), middle (MI), middle-upper (MU) and upper (UP) part of slope, respectively. A finite length of batter pile (10 m) was adopted, and the pile toe was free. Four series of numerical models were proceeded to search the optimum location and inclination angle for pile, one that provided maximum lateral resistance. By analyzing the slope deformation and pile bending moment, the effects of position and inclination angle of pile were determined. The following conclusions could be drawn: (1) The surcharge loading on the slope crest will influence the deformation of slope remarkably. Both the lateral displacement and settlement increases simultaneously with the raise of loading. (2) The lateral displacement and settlement of slope vary as the pile location moves from the crest to the toe. The slope of UP condition has the the minimum lateral displacement, following by MI, MD and MU condition. The ranking of settlement is same with the lateral displacement. That is to say, the UP condition is the best location for piled slope, followed by MI condition. (3) The batter pile is beneficial for slope engineering, which can not only reduce the deformation of slope, but also decrease the bending moment exerted on pile shaft. The inclination angle should be in a reasonable range, a relatively small inclination angle could not fully utilize the potential capacity of batter pile, while exaggerated inclination degree is not befitting for construction. The inclination angle of 15° is an optimal choice for batter pile in slope. (4) The inclination angle has mutual effect with the pile location, they should be considered synchronously. For comprehensive consideration, UP 10°, UP 15°, MI 15° and MI 20° are preferred options for the piled earth slope when subjected to surcharge loading.

All Ion Differential Analysis Refines the Detection of Terminal and Internal Diagnostic Fragment Ions for the Characterization of Biologics Product-Related Variants and Impurities by Middle-down Mass Spectrometry.

Characterization and monitoring of post-translational modifications (PTMs) are key analytical requirements during the development of biologics. Top and middle-down (MD) approaches aim at capturing a direct snapshot of all proteoforms with their combinatorial distribution. However, classical MD data analysis is predominantly limited to the interpretation of terminal ion series and PTMs matched by mass. In this study, time-resolved deconvolution (TRD) maps were produced to detect variants and impurities in Fd, Fc/2, and LC subunits of an IgG1 consistently across multiple samples. Classical MD analysis retrieved terminal ions, suggesting a deamidation at a NN motif for a LC+1 Da species, and inconclusive information for a LC+40 Da species. Additionally, we performed differential analysis of all MS2 ions across unmodified and variant subunit spectra to focus data analysis on spectral differences and reveal diagnostic ions (present, absent, enriched, or depleted ions) before fragment assignment. This sensitive methodology was able to detect diagnostic ions in a chimeric spectrum pointing at a proline-to-histidine sequence variant (+40 Da) missed by classical MD analysis. This methodology was pivotal to unravel relevant terminal ions and internal fragments N-terminal to proline as diagnostic ions to confirm the deamidation site. Moreover, different cleavage propensities were revealed at the deamidated DN site compared to the native NN motif for terminal and internal fragments, which may be tracked as a diagnostic behavior. Differential analysis may refine the detection of novel diagnostic ions and leverage the sequence information on internal fragments for the characterization of product-related variants and impurities by MD mass spectrometry.

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry.

Owing to rapid growth in the elucidation of genome sequences of various organisms, deducing proteome sequences has become imperative, in order to have an improved understanding of biological processes. Since the traditional Edman method was unsuitable for high-throughput sequencing and also for N-terminus modified proteins, mass spectrometry (MS) based methods, mainly based on soft ionization modes: electrospray ionization and matrix-assisted laser desorption/ionization, began to gain significance. MS based methods were adaptable for high-throughput studies and applicable for sequencing N-terminus blocked proteins/peptides too. Consequently, over the last decade a new discipline called ‘proteomics’ has emerged, which encompasses the attributes necessary for high-throughput identification of proteins. ‘Proteomics’ may also be regarded as an offshoot of the classic field, ‘biochemistry’. Many protein sequencing and proteomic investigations were successfully accomplished through MS dependent sequence elucidation of ‘short proteolytic peptides (typically: 7–20 amino acid residues), which is called the ‘shotgun’ or ‘bottom-up (BU)’ approach. While the BU approach continues as a workhorse for proteomics/protein sequencing, attempts to sequence intact proteins without proteolysis, called the ‘top-down (TD)’ approach started, due to ambiguities in the BU approach, e.g., protein inference problem, identification of proteoforms and the discovery of posttranslational modifications (PTMs). The high-throughput TD approach (TD proteomics) is yet in its infancy. Nevertheless, TD characterization of purified intact proteins has been useful for detecting PTMs. With the hope to overcome the pitfalls of BU and TD strategies, another concept called the ‘middle-down (MD)’ approach was put forward. Similar to BU, the MD approach also involves proteolysis, but in a restricted manner, to produce ‘longer’ proteolytic peptides than the ones usually obtained in BU studies, thereby providing better sequence coverage. In this regard, special proteases (OmpT, Sap9, IdeS) have been used, which can cleave proteins to produce longer proteolytic peptides. By reviewing ample evidences currently existing in the literature that is predominantly on PTM characterization of histones and antibodies, herein we highlight salient features of the MD approach. Consequently, we are inclined to claim that the MD concept might have widespread applications in future for various research areas, such as clinical, biopharmaceuticals (including PTM analysis) and even for general/routine characterization of proteins including therapeutic proteins, but not just limited to analysis of histones or antibodies.

Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody.

Pairing light and heavy chains in monoclonal antibodies (mAbs) using top-down (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by high-throughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein, and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LC-MS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.

Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques.

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.