An Essential Role for Alzheimer's-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis.

Human amyloid beta peptide (Aß) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AßOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AßO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aß as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AßOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AßO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AßOs, are linked to Alzheimer's disease (AD). To test whether the AßOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AßO-selective antibody to prematurely lower the levels of AßOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AßOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.

The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

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. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Spectrum of Apolipoprotein AI and Apolipoprotein AII Proteoforms and Their Associations With Indices of Cardiometabolic Health: The CARDIA Study.

Background ApoAI (apolipoproteins AI) and apoAII (apolipoprotein AII) are structural and functional proteins of high-density lipoproteins (HDL) which undergo post-translational modifications at specific residues, creating distinct proteoforms. While specific post-translational modifications have been reported to alter apolipoprotein function, the full spectrum of apoAI and apoAII proteoforms and their associations with cardiometabolic phenotype remains unknown. Herein, we comprehensively characterize apoAI and apoAII proteoforms detectable in serum and their post-translational modifications and quantify their associations with cardiometabolic health indices. Methods and Results Using top-down proteomics (mass-spectrometric analysis of intact proteins), we analyzed paired serum samples from 150 CARDIA (Coronary Artery Risk Development in Young Adults) study participants from year 20 and 25 exams. Measuring 15 apoAI and 9 apoAII proteoforms, 6 of which carried novel post-translational modifications, we quantified associations between percent proteoform abundance and key cardiometabolic indices. Canonical (unmodified) apoAI had inverse associations with HDL cholesterol and HDL-cholesterol efflux, and positive associations with obesity indices (body mass index, waist circumference), and triglycerides, whereas glycated apoAI showed positive associations with serum glucose and diabetes mellitus. Fatty-acid‒modified ApoAI proteoforms had positive associations with HDL cholesterol and efflux, and inverse associations with obesity indices and triglycerides. Truncated and dimerized proteoforms of apoAII were associated with HDL cholesterol (positively) and obesity indices (inversely). Several proteoforms had no significant associations with phenotype. Conclusions Associations between apoAI and AII and cardiometabolic indices are proteoform-specific. These results provide "proof-of-concept" that precise chemical characterization of human apolipoproteins will yield improved insights into the complex pathways through which proteins signify and mediate health and disease.

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. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

HDL modification: recent developments and their relevance to atherosclerotic cardiovascular disease.

PURPOSE OF REVIEW: In the last 2 years, significant advances in the understanding of HDL particle structure and the associations between particle structure, function, and atherosclerosis have been made. We will review and provide clinical and epidemiological context to these recent advances. RECENT FINDINGS: Several recent studies have analyzed the associations between HDL particle size distribution, number, and particle function and specific environmental, behavioral, and pharmacologic exposures. Detailed phenotyping of HDL-associated protein complements, particularly apolipoproteins, strongly suggests structural subspecies of HDL exist with differential associations with HDL function and ASCVD risk. SUMMARY: The recent data on biological and structural variation in HDL suggests the existence of relatively discrete particle species, which share a similar structure and function. We propose that the classical taxonomy that clusters HDL particles by cholesterol content is incomplete. Detailed phenotyping of HDL subspecies in clinical and epidemiological research may yield insights into new risk markers and biochemical pathways that could provide targets for atherosclerotic cardiovascular disease (ASCVD) therapy and prevention in the future.

The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins.

High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.

Top-down characterization of endogenous protein complexes with native proteomics.

Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis.

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

An informatic framework for decoding protein complexes by top-down mass spectrometry.

Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy that supports hierarchical top-down analysis for precise identification and scoring of multi-proteoform complexes by native mass spectrometry.