Adding Color to Mass Spectra of Biopolymers: Charge Determination Analysis (CHARDA) Assigns Charge State to Every Ion Peak.

Traditionally, mass spectrometry (MS) output is the ion abundance plotted versus the ionic mass-to-charge ratio m/z. While employing only commercially available equipment, Charge Determination Analysis (CHARDA) adds a third dimension to MS, estimating for individual peaks their charge states z starting from z = 1 and color coding z in m/z spectra. CHARDA combines the analysis of ion signal decay rates in the time-domain data (transients) in Fourier transform (FT) MS with the interrogation of mass defects (fractional mass) of biopolymers. Being applied to individual isotopic peaks in a complex protein tandem (MS/MS) data set, CHARDA aids peptide mass spectra interpretation by facilitating charge-state deconvolution of large ionic species in crowded regions, estimating z even in the absence of an isotopic distribution (e.g., for monoisotopic mass spectra). CHARDA is fast, robust, and consistent with conventional FTMS and FTMS/MS data acquisition procedures. An effective charge-state resolution Rz ≥ 6 is obtained with the potential for further improvements.

Site-selective template-directed synthesis of antibody Fc conjugates with concomitant ligand release.

Template-directed methods are emerging as some of the most effective means to conjugate payloads at selective sites of monoclonal antibodies (mAbs). We have previously reported a method based on an engineered Fc-III reactive peptide to conjugate a radionuclide chelator to K317 of antibodies with the concomitant release of the Fc-III peptide ligand. Here, our method was redesigned to target two lysines proximal to the Fc-III binding site, K248 and K439. Using energy minimization predictions and a semi-combinatorial synthesis approach, we sampled multiple Fc-III amino acid substituents of A3, H5, L6 and E8, which were then converted into Fc-III reactive conjugates. Middle-down MS/MS subunit analysis of the resulting trastuzumab conjugates revealed that K248 and K439 can be selectively targeted using the Fc-III reactive variants L6Dap, L6Orn, L6Y and A3K or A3hK, respectively. Across all variants tested, L6Orn-carbonate appeared to be the best candidate, yielding a degree and yield of conjugation of almost 2 and 100% for a broad array of payloads including radionuclide chelators, fluorescent dyes, click-chemistry reagents, pre-targeted imaging reagents, and some cytotoxic small molecules. Furthermore, L6Orn carbonate appeared to yield similar conjugation results across multiple IgG subtypes. In vivo proof of concept was achieved by conjugation of NODAGA to the PD1/PD-L1 immune checkpoint inhibitor antibody atezolizumab, followed by PET imaging of PD-L1 expression in mice bearing PD-L1 expressing tumor xenograft using radiolabeled [64Cu]Cu-atezolizumab.

The Antioxidant and Proapoptotic Effects of Sternbergia clusiana Bulb Ethanolic Extract on Triple-Negative and Estrogen-Dependent Breast Cancer Cells In Vitro.

BACKGROUND: Sternbergia clusiana belongs to the Amaryllidaceae family and is recognized for the valuable biological activity of its major bioactive compounds. The aim of the current is to evaluate the anticancer effects of the ethanolic bulb extract of Sternbergia clusiana (ScBEE) on breast cancer cells in vitro and to further reveal the underlying cellular mechanism. METHODS: An MTS cell viability assay was performed on MDA-MB-231 and MCF-7 cells, along with cell cycle analysis, cell death ELISA, Western blot analysis and an ROS production assay to decipher the mechanism of death. LC-MS/MS was also performed to identify the chemical composition of this ethanolic extract. RESULTS: The results show a selective antiproliferative effect on both cell lines with no effect on normal mesenchymal stem cells. Further analysis suggested the activation of the apoptotic pathway as reflected by the increase in cellular and DNA fragmentation and alterations in apoptotic proteins such as Bax, Bcl-2 and c-PARP. ScBEE was also found to exhibit antioxidant effect, as shown by a decrease in ROS production. The underlying mechanism of action was explained by the presence of several bioactive compounds identified by LC-MS/MS, including alkaloids, terpenoids and phenols, which are elaborated in the manuscript. CONCLUSION: This study highlights the antioxidant and anticancerous properties of S.clusiana for breast cancer treatment.

Correction: Tyrosine bioconjugation with hypervalent iodine.

[This corrects the article DOI: 10.1039/D2SC04558C.].

Tyrosine bioconjugation with hypervalent iodine.

Hypervalent iodine reagents have recently emerged as powerful tools for late-stage peptide and protein functionalization. Herein we report a tyrosine bioconjugation methodology for the introduction of hypervalent iodine onto biomolecules under physiological conditions. Tyrosine residues were engaged in a selective addition onto the alkynyl bond of ethynylbenziodoxolones (EBX), resulting in stable vinylbenziodoxolones (VBX) bioconjugates. The methodology was successfully applied to peptides and proteins and tolerated all other nucleophilic residues, with the exception of cysteine. The generated VBX were further functionalized by palladium-catalyzed cross-coupling and azide-alkyne cycloaddition reactions. The method could be successfully used to modify bioactive natural products and native streptavidin to enable thiol-mediated cellular uptake.

High-Throughput Sizing, Counting, and Elemental Analysis of Anisotropic Multimetallic Nanoparticles with Single-Particle Inductively Coupled Plasma Mass Spectrometry.

Nanoparticles (NPs) have wide applications in physical and chemical processes, and their individual properties (e.g., shape, size, and composition) and ensemble properties (e.g., distribution and homogeneity) can significantly affect the performance. However, the extrapolation of information from a single particle to the ensemble remains a challenge due to the lack of suitable techniques. Herein, we report a high-throughput single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS)-based protocol to simultaneously determine the size, count, and elemental makeup of several thousands of (an)isotropic NPs independent of composition, size, shape, and dispersing medium with atomistic precision in a matter of minutes. By introducing highly diluted nebulized aqueous dispersions of NPs directly into the plasma torch of an ICP-MS instrument, individual NPs are atomized and ionized, resulting in ion plumes that can be registered by the mass analyzer. Our proposed protocol includes a phase transfer step for NPs synthesized in organic media, which are otherwise incompatible with ICP-MS instruments, and a modeling tool that extends the measurement of particle morphologies beyond spherical to include cubes, truncated octahedra, and tetrahedra, exemplified by anisotropic Cu NPs. Finally, we demonstrate the versatility of our method by studying the doping of bulk-dilute (<1 at. %) CuAg nanosurface alloys as well as the ease with which ensemble composition distributions of multimetallic NPs (i.e., CuPd and CuPdAg) can be obtained providing different insights in the chemistry of nanomaterials. We believe our combined protocol could deepen the understanding of macroscopic phenomena involving nanoscale structures by bringing about a statistics renaissance in research areas including, among others, materials science, materials chemistry, (nano)physics, (nano)photonics, catalysis, and electrochemistry.

Characterization of the Time-Domain Isotopic Beat Patterns of Monoclonal Antibodies in Fourier Transform Mass Spectrometry.

The time-domain transients in the Fourier transform mass spectrometry (FTMS) analysis of monoclonal antibodies (mAbs) are known to exhibit characteristic isotopic beat patterns. These patterns are defined by the isotopic distributions of all gaseous mAb ions present in the FTMS mass analyzer, originating from single or multiple charge states, and from single or multiple proteoforms. For an isolated charge state of a single proteoform, the mAb isotopic beat pattern resembles narrow splashes of signal amplitude (beats), spaced periodically in the time-domain transient, with broad (often exceeding 1 s) "valleys" between them. Here, we reinforce the importance of isotopic beat patterns for the accurate interpretation and presentation of FTMS data in the analysis of mAbs and other large biopolymers. An updated, mAb-grade version of the transient-mediated FTMS data simulation and visualization tool, FTMS Simulator is introduced and benchmarked. We then apply this tool to evaluate the charge-state dependent characteristics of isotopic beats in mAbs analyses with modern models of Orbitrap and ion cyclotron resonance (ICR) FTMS instruments, including detection of higher-order harmonics. We demonstrate the impact of the isotopic beat patterns on the analytical characteristics of the resulting mass spectra of individual and overlapping mAb proteoforms. The results reported here detail highly nonlinear dependences of resolution and signal-to-noise ratio on the time-domain transient period, absorption or magnitude mode spectra representation, and apodization functions. The provided description and the demonstrated ability to routinely conduct accurate simulations of FTMS data for large biopolymers should aid the end-users of Orbitrap and ICR FTMS instruments in the analysis of mAbs and other biopolymers, including viruses.

Template directed synthesis of antibody Fc conjugates with concomitant ligand release.

Antibodies are an attractive therapeutic modality for cancer treatment as they allow the increase of the treatment response rate and avoid the severe side effects of chemotherapy. Notwithstanding the strong benefit of antibodies, the efficacy of anti-cancer antibodies can dramatically vary among patients and ultimately result in no response to the treatment. Here, we have developed a novel means to regioselectively label the Fc domain of any therapeutic antibody with a radionuclide chelator in a single step chemistry, with the aim to study by SPECT/CT imaging if the radiolabeled antibody is capable of targeting cancer cells in vivo. A Fc-III peptide was used as bait to bring a carbonate electrophilic site linked to a metal chelator and to a carboxyphenyl leaving group in close proximity with an antibody Fc nucleophile amino acid (K317), thereby triggering the covalent linkage of the chelator to the antibody lysine, with the concomitant release of the carboxyphenyl Fc-III ligand. Using CHX-A''-DTPA, we radiolabeled trastuzumab with indium-111 and showed in biodistribution and imaging experiments that the antibody accumulated successfully in the SK-OV-3 xenograft tumour implanted in mice. We found that our methodology leads to homogeneous conjugation of CHX-A''-DTPA to the antibody, and confirmed that the Fc domain can be selectively labeled at K317, with a minor level of unspecific labeling on the Fab domain. The present method can be developed as a clinical diagnostic tool to predict the success of the therapy. Furthermore, our Fc-III one step chemistry concept paves the way to a broad array of other applications in antibody bioengineering.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade.

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs' structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group's contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb's characterization workflows.

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry.

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.

The methylome of the model arbuscular mycorrhizal fungus, Rhizophagus irregularis, shares characteristics with early diverging fungi and Dikarya.

Early-diverging fungi (EDF) are distinct from Dikarya and other eukaryotes, exhibiting high N6-methyldeoxyadenine (6mA) contents, rather than 5-methylcytosine (5mC). As plants transitioned to land the EDF sub-phylum, arbuscular mycorrhizal fungi (AMF; Glomeromycotina) evolved a symbiotic lifestyle with 80% of plant species worldwide. Here we show that these fungi exhibit 5mC and 6mA methylation characteristics that jointly set them apart from other fungi. The model AMF, R. irregularis, evolved very high levels of 5mC and greatly reduced levels of 6mA. However, unlike the Dikarya, 6mA in AMF occurs at symmetrical ApT motifs in genes and is associated with their transcription. 6mA is heterogeneously distributed among nuclei in these coenocytic fungi suggesting functional differences among nuclei. While far fewer genes are regulated by 6mA in the AMF genome than in EDF, most strikingly, 6mA methylation has been specifically retained in genes implicated in components of phosphate regulation; the quintessential hallmark defining this globally important symbiosis.

The pro-apoptotic properties of a phytonutrient rich infusion of A. cherimola leaf extract on AML cells.

Annonaceae family has broad uses in herbal medicine for treatment of several diseases, whether through seeds' or leaves' extracts. The present study investigates the antiproliferative and antitumor activity of Annona cherimola aqueous leaf (AAL) extract/infusion in acute myeloid leukemia (AML) cell lines in vitro. High-resolution LC-MS was first used to analyze the composition of the aqueous extract. Cell proliferation assay, Annexin V staining, cell cycle analysis, dual Annexin V/PI staining, cell death quantification by ELISA, ROS level detection and Western Blotting were then performed to elucidate the therapeutic effects of AAL extract. The results obtained revealed a potent antioxidant activity of AAL extract. Moreover, the extract exhibited dose- and time-dependent antiproliferative effects on AML cell lines by decreasing cell viability with an IC50 of 5.03% (v/v) at 24 h of treatment of KG-1 cells. This decrease in viability was accompanied with a significant increase in apoptotic cell death with cell cycle arrest and flipping of the phosphatidylserine from the inner to the outer leaflet of the cell membrane. The respective overexpression and downregulation of proapoptotic proteins like cleaved caspase-8, cleaved PARP-1 and Bax and antiapoptotic proteins like Bcl-2 further validated the apoptotic pathway induced by AAL on AML cells. Finally, LC-MS revealed the presence of several compounds like fatty acids, terpenes, phenolics, cinnamic acids and flavonoids that could contribute to the antioxidant and anti-cancer effects of this herbal infusion. In addition to the generally known nutritional effects of the Annona cherimola fruit and leaves, the presented data validates the antioxidant and anti-cancerous effects of the leaf infusion on AML cell lines, proposing its potential therapeutic use against acute myeloid leukemia with future in vivo and clinical trials.

Rapid Noninvasive Skin Monitoring by Surface Mass Recording and Data Learning.

Skin problems are often overlooked due to a lack of robust and patient-friendly monitoring tools. Herein, we report a rapid, noninvasive, and high-throughput analytical chemical methodology, aiming at real-time monitoring of skin conditions and early detection of skin disorders. Within this methodology, adhesive sampling and laser desorption ionization mass spectrometry are coordinated to record skin surface molecular mass in minutes. Automated result interpretation is achieved by data learning, using similarity scoring and machine learning algorithms. Feasibility of the methodology has been demonstrated after testing a total of 117 healthy, benign-disordered, or malignant-disordered skins. Remarkably, skin malignancy, using melanoma as a proof of concept, was detected with 100% accuracy already at early stages when the lesions were submillimeter-sized, far beyond the detection limit of most existing noninvasive diagnosis tools. Moreover, the malignancy development over time has also been monitored successfully, showing the potential to predict skin disorder progression. Capable of detecting skin alterations at the molecular level in a nonsurgical and time-saving manner, this analytical chemistry platform is promising to build personalized skin care.

Aom2 S: A new web-based application for DNA/RNA tandem mass spectrometry data interpretation.

RATIONALE: The Analysis of Oligonucleotide Modifications from Mass Spectra (Aom2 S) was created to support the analysis of oligonucleotide mass spectra. This application complements the existing software tools by providing a comprehensive analysis of oligonucleotide fragments from high-resolution tandem mass spectrometry (HR-MS/MS) data in a flexible and user-friendly manner, directly accessible through a web browser without any need for installation. METHODS: MS measurements of aminoC6-DNA and inosine-RNA were performed using an LTQ Orbitrap FT-MS instrument. The obtained data were analyzed by our newly developed open-source package Aom2 S accessible from the ms.epfl.ch web page or directly at https://mstools.epfl.ch/am2s/ to demonstrate the various functionalities of this tool, notably the possibility to identify different product ions from a nucleotide sequence with any fixed/variable modification by matching theoretical isotopic patterns to any experimental mass spectra with similarity scores ranking. RESULTS: A detailed description of the Aom2 S tool with its user-friendly interface is exemplified using HR-MS/MS data of modified DNA and RNA oligonucleotides. Explanations of analysis parameters and tool workflow, as well as multiple options for viewing and exporting the results, are provided. Product ion assignment and modification localization can be achieved in seconds, and results can be exported as tables, matched mass spectra, and fragmentation maps. CONCLUSIONS: A new open source tool (Aom2 S) for the analysis of HR-MS/MS data for modified DNA and RNA oligonucleotides is described. Aom2 S is fast, highly flexible, and versatile, allowing automatic precursor and product ion assignment in a comprehensive manner, including internal fragments and variable modification localization, with clear graphical representation of the results.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Transient-Mediated Simulations of FTMS Isotopic Distributions and Mass Spectra to Guide Experiment Design and Data Analysis.

Fourier transform mass spectrometry (FTMS) applications require accurate analysis of extremely complex mixtures of species in wide mass and charge state ranges. To optimize the related FTMS data analysis accuracy, parameters for data acquisition and the allied data processing should be selected rationally, and their influence on the data analysis outcome is to be understood. To facilitate this selection process and to guide the experiment design and data processing workflows, we implemented the underlying algorithms in a software tool with a graphical user interface, FTMS Isotopic Simulator. This tool computes FTMS data via time-domain data (transient) simulations for user-defined molecular species of interest and FTMS instruments, including diverse Orbitrap FTMS models, followed by user-specified FT processing steps. Herein, we describe implementation and benchmarking of this tool for analysis of a wide range of compounds as well as compare simulated and experimentally generated FTMS data. In particular, we discuss the use of this simulation tool for narrowband, broadband, and low- and high-resolution analysis of small molecules, peptides, and proteins, up to the level of their isotopic fine structures. By demonstrating the allied FT processing artifacts, we raise awareness of a proper selection of FT processing parameters for modern applications of FTMS, including intact mass analysis of proteoforms and top-down proteomics. Overall, the described transient-mediated approach to simulate FTMS data has proven useful for supporting contemporary FTMS applications. We also find its utility in fundamental FTMS studies and creating didactic materials for FTMS teaching.

Modified cyclodextrins as broad-spectrum antivirals.

Viral infections kill millions of people and new antivirals are needed. Nontoxic drugs that irreversibly inhibit viruses (virucidal) are postulated to be ideal. Unfortunately, all virucidal molecules described to date are cytotoxic. We recently developed nontoxic, broad-spectrum virucidal gold nanoparticles. Here, we develop further the concept and describe cyclodextrins, modified with mercaptoundecane sulfonic acids, to mimic heparan sulfates and to provide the key nontoxic virucidal action. We show that the resulting macromolecules are broad-spectrum, biocompatible, and virucidal at micromolar concentrations in vitro against many viruses [including herpes simplex virus (HSV), respiratory syncytial virus (RSV), dengue virus, and Zika virus]. They are effective ex vivo against both laboratory and clinical strains of RSV and HSV-2 in respiratory and vaginal tissue culture models, respectively. Additionally, they are effective when administrated in mice before intravaginal HSV-2 inoculation. Lastly, they pass a mutation resistance test that the currently available anti-HSV drug (acyclovir) fails.

A Self-Assembled Organic/Metal Junction for Water Photo-Oxidation.

We report the in situ self-assembly of TTF, TTF•+, and BF4- or PF6- into p-type semiconductors on the surface of Pt microparticles dispersed in water/acetonitrile mixtures. The visible light photoactivation of these self-assemblies leads to water oxidation forming O2 and H+, with an efficiency of 100% with respect to the initial concentration of TTF•+. TTF•+ is then completely reduced to TTF upon photoreduction with water. The Pt microparticles act as floating microelectrodes whose Fermi level is imposed by the different redox species in solution; here predominantly TTF, TTF•+, and HTTF+, which furthermore showed no signs of decomposition in solution.

Rapid, Selective Extraction of Trace Amounts of Gold from Complex Water Mixtures with a Metal-Organic Framework (MOF)/Polymer Composite.

With the ever-increasing production of electronics, there is an ensuing need for gold extraction from sources other than virgin mines. Currently, there are no technologies reported to date that can effectively and selectively concentrate ultratrace amounts of gold from liquid sources. Here, we provide a blueprint for the design of several highly porous composites made up of a metal-organic framework (MOF) template and redox active, polymeric building blocks. One such composite, Fe-BTC/PpPDA, is shown to rapidly extract trace amounts of gold from several complex water mixtures that include wastewater, fresh water, ocean water, and solutions used to leach gold from electronic waste and sewage sludge ash. The material has an exceptional removal capacity, 934 mg gold/g of composite, and extracts gold from these complex mixtures at record-breaking rates, in as little as 2 min. Further, due to the high cyclability, we demonstrate that the composite can effectively concentrate gold and yield purities of 23.9 K.

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.