Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers.

Individual ion mass spectrometry (IMS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, IMS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. IMS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution.

Improving Top-Down Sequence Coverage with Targeted Fragment Matching.

Top-down mass spectrometry (TDMS) of intact proteins and antibodies enables direct determination of truncations, sequence variants, post-translational modifications, and disulfides without the need for any proteolytic cleavage. While mass deconvolution of top-down tandem mass spectra is typically used to identify fragment masses for matching to candidate proteoforms, larger molecules such as monoclonal antibodies can produce many fragment ions, making spectral interpretation challenging. Here, we explore an alternative approach for proteoform spectral matching that is better suited for larger protein analysis.

Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.

Modern mass spectrometry technology allows for extensive sequencing of the ~ 25 kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits.

Differences in Cardiorespiratory Responses with Three Set-Paced Submaximal Endurance Tests in Community-Dwelling Older Adults.

A variety of submaximal exercise tests are commonly used in clinical practice to determine an individual's exercise capacity and cardiorespiratory fitness. This study explored differences in cardiorespiratory and perceived exertion responses following the completion of three set-paced exercise tests. A prospective, observational, cross-sectional design assessed 30 healthy communityd-welling older adults, who participated in three submaximal exercise tests, including seated marching (SM), standing marching (STM), and standing stepping (STS).

Dyadic investigations of past traumatic events and affectionate touch frequency in couples.

Past traumatic events negatively affect romantic relationships, yet their impact on affectionate touch, an important predictor of psychological and relational well-being, remains unknown. In two preregistered studies with nonclinical samples, we hypothesized that traumatic events are negatively associated with affectionate touch frequency for both the victim (i.e.

Filling the gaps in peptide maps with a platform assay for top-down characterization of purified protein samples.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms.

Individualizing the LIFE Curriculum to Establish Flexible Cooking Skills in a Neurodivergent Young Adult.

The present study used a multiple baseline across skills design to evaluate use of an adapted LIFE skills curriculum (Dixon, 2021) to teach various cooking tasks to a young adult diagnosed with ASD, schizophrenia, and bipolar disorder. The participant learned and completed recipes and related vocational tasks. Results showed efficient task acquisition for all recipes and maintenance of vocational tasks during follow-up probes.

Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring.

Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics, termed proteoform reaction monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics, such as sensitivity and reproducibility.

Automated imaging and identification of proteoforms directly from ovarian cancer tissue.

The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based multiplexed workflow for top-down tandem MS (MS) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples.

Advancing Intact Protein Quantitation with Updated Deconvolution Routines.

Analysis of intact proteins by mass spectrometry enables direct quantitation of the specific proteoforms present in a sample and is an increasingly important tool for biopharmaceutical and academic research. Interpreting and quantifying intact protein species from mass spectra typically involves many challenges including mass deconvolution and peak processing as well as determining optimal spectral averaging parameters and matching masses to theoretical proteoforms. Each of these steps can present informatic hurdles, as parameters often need to be tailored specifically to the data sets.

Native top-down proteomics reveals EGFR-ERα signaling crosstalk in breast cancer cells dissociates NUTF2 dimers to modulate ERα signaling and cell growth.

Oligomerization of proteins and their modified forms (proteoforms) produces functional protein complexes . Complexoforms are complexes that consist of the same set of proteins with different proteoforms . The ability to characterize these assemblies within cells is critical to understanding the molecular mechanisms involved in disease and to designing effective drugs.

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS).

High-Throughput Deconvolution of Intact Protein Mass Spectra for the Screening of Covalent Inhibitors.

Deconvolution from intact protein mass-to-charge spectra to mass spectra is essential to generate interpretable data for mass spectrometry (MS) platforms coupled to ionization sources that produce multiply charged species. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) can be used to analyze intact proteins in multiwell microtiter plates with speed matching small molecule analyses (at least 1 Hz). However, the lack of compatible deconvolution software has limited its use in high-throughput screening applications.

Breastfeeding, Community Vulnerability, Resilience, and Disasters: A Snapshot of the United States Gulf Coast.

Climate change-induced disasters are increasing in intensity and frequency in the United States. Infant feeding in the aftermath of an extreme event is particularly challenging, especially given large variations in community vulnerability and resilience. The aim of this study was to identify the physical, social, and spatial vulnerabilities of communities along the Gulf Coast and highlight locations where high (or low) breastfeeding initiation rates have the potential to offset (or exacerbate) infant feeding challenges in the wake of a disaster.

Evaluating an ACT-Based Brief Intervention for Educators Treatment Package on Reported Well-Being and ACT-Consistent Language in the Classroom.

Educators continue to experience stress and burnout, both of which have been negatively impacted by the COVID-19 pandemic, and there continues to be a need to develop interventions that support not only educators' well-being, but a climate within school buildings that fosters psychological well-being for students and school staff alike. Acceptance and Commitment Therapy (ACT) is one promising approach to interventions for both educator and student psychological well-being. The present study sought to evaluate the effect of a low-dosage, online, and remotely delivered ACT intervention for educators on self-reported burnout, psychological flexibility, ACT knowledge, and frequency of use of ACT-consistent language while teaching in an alternative educational setting.

ProSight Annotator: Complete control and customization of protein entries in UniProt XML files.

The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms.

Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode.

The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies.