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.

Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-Down Proteomics.

The dynamic range challenge for the detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundance proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between ∼1 μg/mL and ∼10 pg/mL in absolute abundance, providing up to a 10-fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 μg/mL) were detected.

Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms.

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (IMS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent IMS acquisition.

Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-down Proteomics.

The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between ∼1 µg/mL and ∼10 pg/mL in absolute abundance, providing up to 10 -fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 µg/mL) were detected.

Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra.

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.

Identification of Splice Variants and Isoforms in Transcriptomics and Proteomics.

Alternative splicing is pivotal to the regulation of gene expression and protein diversity in eukaryotic cells. The detection of alternative splicing events requires specific omics technologies. Although short-read RNA sequencing has successfully supported a plethora of investigations on alternative splicing, the emerging technologies of long-read RNA sequencing and top-down mass spectrometry open new opportunities to identify alternative splicing and protein isoforms with less ambiguity.

Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS.

SARS-CoV-2 Omicron (B.1.1.

The Human Proteoform Atlas: a FAIR community resource for experimentally derived proteoforms.

The Human Proteoform Atlas (HPfA) is a web-based repository of experimentally verified human proteoforms on-line at http://human-proteoform-atlas.org and is a direct descendant of the Consortium of Top-Down Proteomics' (CTDP) Proteoform Atlas. Proteoforms are the specific forms of protein molecules expressed by our cells and include the unique combination of post-translational modifications (PTMs), alternative splicing and other sources of variation deriving from a specific gene.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells.

Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label.

Unlabelled: Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron-electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to 'knock out' superfluous cysteine residues.

Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells.

Interaction of oxidative stress and neurotrauma in ALDH2 mice causes significant and persistent behavioral and pro-inflammatory effects in a tractable model of mild traumatic brain injury.

Oxidative stress induced by lipid peroxidation products (LPP) accompanies aging and has been hypothesized to exacerbate the secondary cascade in traumatic brain injury (TBI). Increased oxidative stress is a contributor to loss of neural reserve that defines the ability to maintain healthy cognitive function despite the accumulation of neuropathology. ALDH2 mice are unable to clear aldehyde LPP by mitochondrial aldehyde dehydrogenase-2 (Aldh2) detoxification and provide a model to study mild TBI (mTBI), therapeutic interventions, and underlying mechanisms.

Design and Synthesis of Basic Selective Estrogen Receptor Degraders for Endocrine Therapy Resistant Breast Cancer.

The clinical steroidal selective estrogen receptor (ER) degrader (SERD), fulvestrant, is effective in metastatic breast cancer, but limited by poor pharmacokinetics, prompting the development of orally bioavailable, nonsteroidal SERDs, currently in clinical trials. These trials address local breast cancer as well as peripheral metastases, but patients with brain metastases are generally excluded because of the lack of blood-brain barrier penetration. A novel family of benzothiophene SERDs with a basic amino side arm (B-SERDs) was synthesized.

Pharmacological manipulation of cGMP and NO/cGMP in CNS drug discovery.

The development of small molecule modulators of NO/cGMP signaling for use in the CNS has lagged far behind the use of such clinical agents in the periphery, despite the central role played by NO/cGMP in learning and memory, and the substantial evidence that this signaling pathway is perturbed in neurodegenerative disorders, including Alzheimer's disease. The NO-chimeras, NMZ and Nitrosynapsin, have yielded beneficial and disease-modifying responses in multiple preclinical animal models, acting on GABA and NMDA receptors, respectively, providing additional mechanisms of action relevant to synaptic and neuronal dysfunction. Several inhibitors of cGMP-specific phosphodiesterases (PDE) have replicated some of the actions of these NO-chimeras in the CNS.

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines.

A Bifunctional Spin Label for Ligand Recognition on Surfaces.

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin-labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin-labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported.

New developments in spin labels for pulsed dipolar EPR.

Spin labelling is a chemical technique that enables the integration of a molecule containing an unpaired electron into another framework for study. Given the need to understand the structure, dynamics, and conformational changes of biomacromolecules, spin labelling provides a relatively non-intrusive technique and has certain advantages over X-ray crystallography; which requires high quality crystals. The technique relies on the design of binding probes that target a functional group, for example, the thiol group of a cysteine residue within a protein.