Biology subjects

Van Eyk, J. E.

Publications and source records attributed to Van Eyk, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Joining Two Perspectives of the Proteome with the Trypsin+HTA Protease Workflow

Proteolysis can be completed in a single-step, rapid (5-30 minutes) reaction using hyperthermoacidic archaeal (HTA) proteases. Protocol simplicity and stability of HTA proteases in ambient storage conditions removes many logistical barriers to widespread deployment of proteomic analysis. HTA protease cleavage selectivity is complicated but quantitatively reproducible and generates peptides that negligibly overlap with trypsin. Tryptic and HTA protease peptides emphasize different proteins and provide complementary coverage of amino acid sequence and post-translational modification (PTM) sites. Digestion of a sample's separate aliquot with HTA protease is a minor addition to trypsin-based workflows, but LC-MS analysis of both sets of peptides is not practical for large cohorts. This manuscript evaluates the identifications and reproducibility of LC-MS analysis of tryptic and HTA protease peptide mixtures. Adding HTA protease peptides to 20% increased total protein identifications by 24% on average in neat plasma and identified 216 additional non-artifact modifications compared to conventional trypsin-only bottom-up proteomic analysis. This approach is reproducible and does not increase sample preparation or analysis time. Peptides from each protease present independent opportunities to quantify overlapping proteins and a quantification model was developed in which the protease with the most complete data is selected for each protein while the second protease is used to infer missing values. This cross-protease inference is more accurate than the alternative minimum, mean, or nearest neighbor imputations. Finally, biomarkers discovered through HTA protease peptides are amenable to rapid HTA protease only proteomic workflows thus streamlining translation from discovery to fieldable applications.

biochemistry↗

bioRxiv2026

Gene therapy targeting of AKAP6β-CaMKII signalosomes improves myocardial inflammation and heart failure in a swine model of cardiometabolic syndrome

BackgroundCardiometabolic heart failure with preserved ejection fraction (HFpEF) is associated with systemic and cardiac inflammation and diastolic dysfunction. A-kinase anchoring protein 6{beta} (AKAP6{beta}) is a scaffold protein located at the cardiomyocyte outer nuclear membrane that promotes pathological cardiac remodeling via the recruitment of multiple regulatory proteins including protein kinases. In mice, adeno-associated virus (AAV) mediated expression of a peptide based upon a kinase binding domain (KBD) within AKAP6{beta} inhibited the development of heart failure due to chronic pressure overload. Whether KBD expression can also inhibit the development of cardiometabolic heart failure is unknown, and if so, the mechanism of KBD action in HFpEF has yet to be explored. MethodsThe efficacy of a cardiotropic self-complementary AAV gene therapy that expresses the AKAP6{beta} KBD peptide (AAV9sc.KBD) was tested in a female Ossabaw swine model of cardiometabolic syndrome and HFpEF. Single nucleus and bulk RNA sequencing of swine heart tissue and immunoprecipitation-mass spectrometry, live cell imaging, and biochemical assays using primary rat cardiomyocytes were employed to study KBD mechanism of action. ResultsAAV9sc.KBD inhibited the development of diastolic dysfunction and heart failure in the Ossabaw model, without negatively impacting systolic function. The improvement in cardiac phenotype was associated with decreased T-cell myocardial infiltrates and partial reversal of pathological gene expression. An unbiased interactome study revealed that the KBD peptide binds Ca2+/calmodulin-dependent protein kinase II (CaMKII), identifying CaMKII as a new AKAP6{beta} binding partner. Perinuclear CaMKII activity detected by live cell imaging required AKAP6{beta} expression and was inhibited by KBD expression. In addition, the CaMKII substrate Inhibitor of NF-{kappa}B Kinase {beta} (IKK{beta}) bound AKAP6{beta}. IKK phosphorylation in the Ossabaw model and in myocytes was inhibited by KBD expression, and NF-{kappa}B nuclear translocation in myocytes was dependent upon AKAP6{beta}-CaMKII protein complex formation. AAV9sc.KBD treatment inhibited cardiomyocyte NF-{kappa}B-dependent gene expression in the Ossabaw model. ConclusionsRegulated by perinuclear AKAP6{beta}-CaMKII signalosomes, NF-{kappa}B pro-inflammatory gene expression in cardiomyocytes participates in a positive feedback loop with cardiac inflammation promoting HFpEF. Proof-of-concept is provided in a large animal model that gene therapy-based cardiomyocyte expression of the KBD peptide will prevent cardiac dysfunction in cardiometabolic syndrome. Clinical PerspectiveO_ST_ABSWhat is newC_ST_ABSO_LIThe cardiomyocyte-selective gene therapy AAV9sc.KBD, which targets signalosomes organized by the scaffold protein AKAP6{beta}, is shown to inhibit myocardial T-cell infiltration and improve cardiac structure and function in a large animal model of cardiometabolic HFpEF. C_LIO_LIThe AKAP6{beta} KBD peptide is shown to bind and inhibit the function of CaMKII. C_LIO_LICaMKII and IKK{beta} are shown to participate in perinuclear AKAP6{beta} signalosomes, where they regulate activation of the NF-{kappa}B pro-inflammatory gene regulatory pathway. C_LI Clinical implicationsO_LIProof-of-concept for a novel strategy for the treatment of HFpEF is provided, intracellular expression by a cardiomyocyte-selective gene therapy vector of an inhibitory peptide, which will inhibit compartmentalized intracellular signal transduction. C_LIO_LIIn conjunction with previous studies in small rodents, the new data obtained in Ossabaw swine support clinical translation of the AAV9sc.KBD gene therapy. C_LI

physiology↗

bioRxiv2026

Expanded Proteome Coverage Powered by Advanced Ion Processing Enables Deep Single-Cell Drug Response Subtyping in Human Stem Cell Derived Cardiomyocytes

Single-cell proteomics (SCP) enables the study of cellular heterogeneity at the functional level but remains limited by incomplete proteome coverage and high data missingness. Here, we present an enhanced label-free SCP workflow that leverages the timsUltra AIP mass spectrometry platform equipped with the Athena Ion Processor (AIP). Across a controlled dilution series of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), AIP-enabled acquisition consistently increased proteome depth and detection consistency across cells at all input levels. In single iPSC-CMs, the timsUltra AIP quantified up to 3,858 protein groups, averaging [~]1,300 proteins per cell, enabling robust proteome-level classification of cardiomyocyte subtypes. Using a reference-based protein classifier, cells were stratified into mature cardiomyocytes and less differentiated cell states, revealing substantial baseline heterogeneity. Importantly, increased single-cell sensitivity translated directly into biological insight, as approximately 30% of differentially expressed proteins associated with subtype-specific drug responses were detected exclusively by timsUltra AIP. Application of this workflow to PR-364 (a mitophagy boosting drug) dose-response experiment uncovered distinct, subtype-dependent pathway adaptations. Mature cardiomyocytes exhibited dose-dependent increases in mitochondrial and metabolic pathway activity, while immature cells showed enrichment of cytoskeletal and developmental programs. These effects were partially obscured in simulated bulk analyses, highlighting the value of single-cell resolution. Together, these results demonstrate that improved fragment ion transmission and utilization translate directly into enhanced biological insight, enabling more comprehensive and functionally relevant single-cell proteomics.

molecular biology↗

bioRxiv2025

Multicenter HFpEF study identifies sex disparity linked with two discrete cardiac proteomic signatures

Heart failure with preserved ejection fraction (HFpEF) is epidemic, with an incidence exceeding that of heart failure with reduced ejection fraction (HFrEF). Sex differences in HFpEF phenotype have been observed, particularly linked to aging and metabolic comorbidities in women. Here, we report a multi-centre proteomic analysis of HFpEF and HFrEF cardiac samples. Using tissues and clinical data derived from multiple institutional biobanks, in-depth characterization of the proteome of ventricular samples was undertaken. Relative to HFrEF, the HFpEF cohort exhibited minimal proteome composition overlap and pronounced heterogeneity. A key finding of this investigation is that sex per se did not confer a distinctive proteomic HFpEF signature. Rather, two HFpEF proteomic profiles were identified, differing significantly in sex ratios. The identification of two Clusters revealed that HFpEF has two predominant proteomic signatures at the molecular level, marked by differences in the extent and nature of structural and contractile machinery and by local cellular and ECM communications. Upstream regulator analysis identified various molecular leads to be pursued in defining these two HFpEF profiles. Our findings offer specific opportunities for new exploration of therapeutic options to target the spectrum of HFpEF proteomic diversity and identify potential drug targeting prospects.

molecular biology↗

bioRxiv2025

Development and Clinical Evaluation of a Multiplexed Health Surveillance Panel Using Ultra High-Throughput PRM-MS in an Inflammatory Bowel Disease Cohort.

Despite advances in clinical proteomics, translating protein biomarker discoveries into clinical use remains challenging due to the technical complexity of the validation process. Targeted MS-based proteomics approaches such as parallel reaction monitoring (PRM) offer sensitive and specific assays for biomarker translation. In this study, we developed a multiplex PRM assay using the Stellar mass spectrometry platform to quantify 57 plasma proteins, including 21 FDA-approved proteins. Loading curves (11-points) were performed at 4 sample throughputs (100, 144, 180, and 300 samples per day) using independent, optimized, and scheduled PRM methods. Following optimization, an inflammatory bowel disease (IBD) cohort of plasma samples (493 IBD, 509 matched controls) was analyzed at a throughput of 180 SPD. To monitor system performance, the study also included 1,000 additional injections for system suitability tests, low-, middle-, and high-quality controls, washes, and blanks. Using this approach, we observed high quantifiability (linearity, sensitivity, reproducibility) in the PRM assay and consistent in data acquisition across a large cohort. We also validated the candidate IBD markers, C-reactive protein and orosomucoid protein, identified in a recent discovery experiment.

biochemistry↗

bioRxiv2025

A targeted LC MS/MS assay of a health surveillance panel and its application to chronic kidney disease

Robust and reproducible assays capable of specific and quantitative monitoring of multiple biologically important proteins amongst the thousands of human plasma proteins can potentially be used to distinguish health versus disease. In this study, we established an LC-MS assay to monitor a Health Surveillance Panel (HSP) comprising 60 circulating plasma proteins selected based on their broad biological functions and assay performance. Plasma samples were prepared for proteomic analysis in an automated process. A scheduled LC-MRM assay with a 30-minute 5% - 35% acetonitrile gradient and 50.5 minutes of total run time was used to quantify the 60 endogenous proteins by monitoring 364 transitions from 117 proteotypic peptides along with their stable isotopic labeled standard peptides in a single assay. For each proteotypic peptide, we selected a quantifier ion and at least two qualifier ions. The quantifier ions have a linear response over a 100-fold range, and the peak area ratios of the three peptide ions were consistent. As proof of concept, we evaluated the performance of our HSP assay in a case-control study of progressive chronic kidney disease (CKD). Reduced plasma concentrations of alpha-2-antiplasmin, antithrombin-III, and immunoglobulin heavy constant alpha 1 correlated with CKD indicated by reduced GFR with p values < 0.05. These results demonstrate that the HSP proteins can be accurately and reproducibly quantified with a high-quality multiplexed MRM assay and the HSP assay can detect disease-associated differences.

biochemistry↗

bioRxiv2024

Targeted glycophagy ATG8 therapy for diabetic heart disease

Diabetic heart disease is highly prevalent and is associated with the early development of impaired diastolic relaxation. The mechanisms of diabetic heart disease are poorly understood and it is a condition for which there are no targeted therapies. Recently, disrupted glycogen-autophagy (glycophagy) and glycogen accumulation have been identified in the diabetic heart. Glycophagy involves glycogen receptor binding and linking with an ATG8 protein to locate and degrade glycogen within an intracellular phago-lysosome. Here we show that glycogen receptor protein STBD1 (starch-binding-domain-protein-1) is mobilized early in the cardiac glycogen response to metabolic challenge in vivo, and that deficiency of a specific ATG8 family protein, Gabarapl1 ({gamma}-aminobutyric-acid-receptor-associated-protein-like-1) is associated with diastolic dysfunction in diabetes. Gabarapl1 gene delivery treatment remediated cardiomyocyte and cardiac diastolic dysfunction in type 2 diabetic mice and diastolic performance of diabetic human iPSC-derived cardiac organoids. We identify glycophagy dysregulation as a mechanism and potential treatment target for diabetic heart disease.

physiology↗

bioRxiv2024

Blood to Biomarker Quantitation in Under One Hour with Rapid Proteomics using a Hyperthermoacidic Protease

Multi-step multi-hour tryptic proteolysis has limited the utility of bottom-up proteomics for cases that require immediate quantitative information. The recently available hyperthermoacidic (HTA) protease "Krakatoa" digests samples in a single 5 to 30-minute step at pH 3 and >80 {degrees}C; conditions that disrupt most cells and tissues, denature proteins, and block disulfide reformation. The combination of quick single-step sample preparation with high throughput dual trapping column single analytical column (DTSC) liquid chromatography-mass spectrometry (LC-MS) achieves "Rapid Proteomics" in which the time from sample collection to actionable data is less than 1 hour. The presented development and systematic evaluation of this methodology found reproducible quantitation of over 160 proteins from just 1 microliter of whole blood. Furthermore, the preference of the HTA-protease for intact proteins over peptides allows for sensitive targeted quantitation of the Angiotensin I and II bioactive peptides in under half an hour. With these methods we analyzed serum and plasma from 53 individuals and quantified Angiotensin and proteins that were not detected with trypsin. This assessment of Rapid Proteomics suggests that concentration of circulating protein and peptide biomarkers could be measured in almost real-time by LC-MS. TOC FigureRapid proteomics enables near real-time monitoring of circulating blood biomarkers. One microliter of blood is collected every 8 minutes, digested for 20 minutes, and then analyzed by targeted mass spectrometry for 8 minutes. This results in a 30-minute delay with datapoints every 8 minutes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/596979v1_figTOC1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1bb0dd8org.highwire.dtl.DTLVardef@189e3dforg.highwire.dtl.DTLVardef@1cf4a5dorg.highwire.dtl.DTLVardef@1b3e58_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers [~]90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types. Graphical abstract created with biorender.com O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/591396v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1cba4c1org.highwire.dtl.DTLVardef@adb231org.highwire.dtl.DTLVardef@18db263org.highwire.dtl.DTLVardef@1babde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Paradigm shift in biomarker translation: a pipeline to generate clinical grade biomarker candidates from DIA-MS discovery

Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package. This innovative tool uses the discovery cohort analysis to select precursors, peptides, and proteins that adhere to established targeted assay criteria. TEAQ was applied to Data-Independent Acquisition MS data from plasma samples acquired on an Orbitrap Astral MS. Identified precursors were evaluated for linearity, specificity, repeatability, reproducibility, and intra-protein correlation from 11-point loading curves under three throughputs, to develop a resource for clinical-grade targeted assays. From a clinical cohort of individuals with inflammatory bowel disease (n=492), TEAQ successfully identified 1116 signature peptides for 327 quantifiable proteins from 1180 identified proteins. Embedding stringent selection criteria adaptable to targeted assay development into the analysis of discovery data will streamline the transition to validation and clinical studies.

biochemistry↗

bioRxiv2024

Mechanical loading reveals an intrinsic cardiomyocyte stiffness contribution to diastolic dysfunction in murine cardiometabolic disease

Cardiometabolic syndromes including diabetes and obesity are associated with occurrence of heart failure with diastolic dysfunction. There are no specific treatments for diastolic dysfunction, and therapies to manage symptoms have limited efficacy. Understanding of the cardiomyocyte origins of diastolic dysfunction is an important priority to identify new therapeutics. The investigative goal was to experimentally define in vitro stiffness properties of isolated cardiomyocytes derived from rodent hearts exhibiting diastolic dysfunction in vivo in response to dietary induction of cardiometabolic disease. Male mice fed a high fat/sugar diet (HFSD vs control) exhibited diastolic dysfunction (echo E/e doppler ratio). Intact paced cardiomyocytes were functionally investigated in three conditions: non-loaded, loaded and stretched. Mean stiffness of HFSD cardiomyocytes was 70% higher than control. E/e for the origin hearts was elevated by 35%. A significant relationship was identified between in vitro cardiomyocyte stiffness and in vivo dysfunction severity. With conversion from non-loaded to loaded condition, the decrement in maximal sarcomere lengthening rate was more accentuated in HFSD cardiomyocytes (vs control). With stretch, the Ca2+ transient decay time course was prolonged. With increased pacing, cardiomyocyte stiffness was elevated, yet diastolic Ca2+ elevation was attenuated. Our findings show unequivocally that cardiomyocyte mechanical dysfunction cannot be detected by analysis of non-loaded shortening. Collectively, these findings demonstrate that a component of cardiac diastolic dysfunction in cardiometabolic disease is derived from cardiomyocyte stiffness. Differential responses to load, stretch and pacing suggest that a previously undescribed alteration in myofilament-Ca2+ interaction contributes to intrinsic cardiomyocyte stiffness in cardiometabolic disease. KEY POINTSO_LIUnderstanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. C_LIO_LIIn this study cardiac function was measured in vivo (echocardiography) for mice fed a high-fat/sugar diet (HFSD, [≥]25weeks). Performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions in vitro. C_LIO_LICalibrated cardiomyocyte stretches demonstrated that stiffness (stress/strain) was elevated in HFSD cardiomyocytes in vitro and correlated with diastolic dysfunction (E/e) in vivo. HFSD cardiomyocyte Ca2+ transient decay was prolonged in response to stretch. Stiffness was accentuated with pacing increase while the elevation in diastolic Ca2+ was attenuated. C_LIO_LIData show unequivocally that cardiomyocyte mechanical dysfunction cannot be detected by analysis of non-loaded shortening. C_LIO_LIThese findings suggest that stretch-dependent augmentation of the myofilament-Ca2+ response during diastole partially underlies elevated cardiomyocyte stiffness and diastolic dysfunction of hearts of animals with cardiometabolic disease. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/581448v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1a2fd4corg.highwire.dtl.DTLVardef@1a398ceorg.highwire.dtl.DTLVardef@184ff46org.highwire.dtl.DTLVardef@936923_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure LegendC_FLOATNO Understanding cardiomyocyte stiffness components is an important priority for identifying new therapeutics for diastolic dysfunction, a key feature of cardiometabolic disease. In this study cardiac function was measured in vivo (echocardiography) for mice fed a high-fat/sugar diet (HFSD, [≥]25weeks). Performance of intact isolated cardiomyocytes derived from the same hearts was measured during pacing under non-loaded, loaded and stretched conditions in vitro. Calibrated cardiomyocyte stretches demonstrated that stiffness was elevated in HFSD cardiomyocytes in vitro and correlated with diastolic dysfunction (E/e) in vivo. These findings show that stiff hearts are characterized by stiff cardiomyocytes in metabolic disease. C_FIG

physiology↗

bioRxiv2023

Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics

BackgroundThe wide dynamic range of circulating proteins coupled with the diversity of proteoforms present in plasma has historically impeded comprehensive and quantitative characterization of the plasma proteome at scale. Automated nanoparticle (NP) protein corona-based proteomics workflows can efficiently compress the dynamic range of protein abundances into a mass spectrometry (MS)-accessible detection range. This enhances the depth and scalability of quantitative MS-based methods, which can elucidate the molecular mechanisms of biological processes, discover new protein biomarkers, and improve comprehensiveness of MS-based diagnostics. MethodsInvestigating multi-species spike-in experiments and a cohort, we investigated fold-change accuracy, linearity, precision, and statistical power for the using the Proteograph Product Suite, a deep plasma proteomics workflow, in conjunction with multiple MS instruments. ResultsWe show that NP-based workflows enable accurate identification (false discovery rate of 1%) of more than 6,000 proteins from plasma (Orbitrap Astral) and, compared to a gold standard neat plasma workflow that is limited to the detection of hundreds of plasma proteins, facilitate quantification of more proteins with accurate fold-changes, high linearity, and precision. Furthermore, we demonstrate high statistical power for the discovery of biomarkers in small- and large-scale cohorts. ConclusionsThe automated NP workflow enables high-throughput, deep, and quantitative plasma proteomics investigation with sufficient power to discover new biomarker signatures with a peptide level resolution.

biochemistry↗

bioRxiv2023

ABDS: tool suite for analyzing biologically diverse samples

MotivationAnalytics tools are essential to identify informative molecular features about different phenotypic groups. Among the most fundamental tasks are missing value imputation, signature gene detection, and expression pattern visualization. However, most commonly used analytics tools may be problematic for characterizing biologically diverse samples when either signature genes possess uneven missing rates across different groups yet involving complex missing mechanisms, or multiple biological groups are simultaneously compared and visualized. ResultsWe develop ABDS tool suite tailored specifically to analyzing biologically diverse samples. Mechanism-integrated group-wise imputation is developed to recruit signature genes involving informative missingness, cosine-based one-sample test is extended to detect enumerated signature genes, and unified heatmap is designed to comparably display complex expression patterns. We discuss the methodological principles and demonstrate the conceptual advantages of the three software tools. We also showcase the biomedical applications of these individual tools. Implemented in open-source R scripts, ABDS tool suite complements rather than replaces the existing tools and will allow biologists to more accurately detect interpretable molecular signals among diverse phenotypic samples. Availability and implementationThe R Scripts of ABDS tool suite is freely available at https://github.com/niccolodpdu/ABDS. Contactyuewang@vt.edu Supplementary informationSupplementary materials are available at Bioinformatics Advances online.

bioinformatics↗

bioRxiv2023

Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious requiring two hours of mass spectrometry time per single muscle fiber; 50 fibers would take approximately four days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 minutes total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 hours. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Sixty-five proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, muscle structure and regulation. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.

biochemistry↗

bioRxiv2022

High Field Asymmetric Waveform Ion Mobility Spectrometry- Mass Spectrometry to Enhance Cardiac Muscle Proteome Coverage

Heart tissue sample preparation for mass spectrometry (MS) analysis that includes pre-fractionation reduces the cellular protein dynamic range and increases the relative abundance of non-sarcomeric proteins. We previously described "IN-Sequence" (IN-Seq) where heart tissue lysate is sequentially partitioned into three subcellular fractions to increase the proteome coverage than a single direct tissue analysis by mass spectrometry. Here, we report an adaptation of the high-field asymmetric ion mobility spectrometry (FAIMS) coupled to mass spectrometry, and the establishment of a simple one step sample preparation coupled with gas-phase fractionation. FAIMS approach substantially reduces manual sample handling, significantly shortens MS instrument processing time, and produces unique protein identification and quantification approximating the commonly used IN-Seq method in for less time requirement.

molecular biology↗

bioRxiv2022

Parallelization with Dual-Trap Single-Column Configuration Maximizes Throughput of Proteomic Analysis

Proteomic analysis on the scale that captures population and biological heterogeneity over hundreds to thousands of samples requires rapid mass spectrometry methods which maximize instrument utilization (IU) and proteome coverage while maintaining precise and reproducible quantification. To achieve this, a short liquid chromatography gradient paired to rapid mass spectrometry data acquisition can be used to reproducibly profile a moderate set of analytes. High throughput profiling at a limited depth is becoming an increasingly utilized strategy for tackling large sample sets but the time spent on loading the sample, flushing the column(s), and re-equilibrating the system reduces the ratio of meaningful data acquired to total operation time and IU. The dual-trap single-column configuration presented here maximizes IU in rapid analysis (15 min per sample) of blood and cell lysates by parallelizing trap column cleaning and sample loading and desalting with analysis of the previous sample. We achieved 90% IU in low micro-flow (9.5 {micro}L/min) analysis of blood while reproducibly quantifying 300-400 proteins and over 6,000 precursor ions. The same IU was achieved for cell lysates, in which over 4,000 proteins (3,000 at CV below 20%) and 40,000 precursor ions were quantified at a rate of 15 minutes/sample. Thus, deployment of this dual-trap single column configuration enables high throughput epidemiological blood-based biomarker cohort studies and cell-based perturbation screening.

biochemistry↗

bioRxiv2021

A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes

BackgroundActivation of stress pathways intrinsic to the {beta} cell are thought to both accelerate {beta} cell death and increase {beta} cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking. MethodsTo identify temporal and disease-related changes in islet {beta} cell protein expression, data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. FindingsIn islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of {beta} cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, {beta} cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls. InterpretationWe identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of {beta} cell stress in T1D.

physiology↗

bioRxiv2021

Mechanistic dissection of global proteomic changes in rats with heart failure and preserved ejection fraction

IntroductionHeart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, pulmonary congestion and exercise intolerance. Previous preclinical studies show that treatment with cardiosphere-derived cells (CDCs) improves diastolic function, attenuates arrhythmias and prolongs survival in a rat HFpEF model. Here we characterize the myocardial proteome and diastolic function in HFpEF, with and without CDC therapy. As an initial strategy for identifying pathways worthy of further mechanistic dissection, we correlated CDC-responsive proteomic changes with functional improvements. Methods and ResultsDahl salt-sensitive rats fed high-salt diet, with verified diastolic dysfunction, were randomly assigned to intracoronary CDCs or placebo. Dahl rats fed a low salt diet served as controls. Phenotyping was by echocardiography (E/A ratio) and invasive hemodynamic monitoring (time constant of relaxation Tau, and left ventricular end-diastolic pressure [LVEDP]). CDC treatment improved diastolic function as indicated by a normalized E/A ratio, a 33.3% reduction in Tau, and a 47% reduction of LVEDP. Mass spectrometry of left ventricular tissues (n=6/group) revealed changes in transcription and translation pathways in this rat HFpEF model and was also recapitulated in human HFpEF. These pathways were enhanced following CDC treatment in the animal model (205 proteins and 32 phosphorylated residues accounting for 37% and 19% of all changes, respectively). Among all CDC-sensitive pathways, 65% can be linked to at least 1 of 7 upstream regulators, among which several are of potential relevance for regulating protein expression. To probe newly-synthesized proteins AHA labeling was carried out in isolated rat cardiomyocytes obtained from HFpEF groups, with and without CDC therapy. Five of the initial upstream regulators (HNF4A, MTOR, MYC, TGF{beta}1, and TP53) were linked to proteins expressed exclusively (or increased) with CDC treatment. All 32 phosphorylated residues of proteins involved in transcription/translation altered specifically by CDC treatment had predicted kinases (Protein kinase C (PKC) being the most dominant) and known to be regulated by MYC, TGF{beta}1 and/or TP53. Western blot analysis of those 5 upstream regulators showed that TGF{beta}1, TP53, and Myc were significantly decreased in LV from CDC treated animals, whereas MTOR and HNF4A showed a significant increase compared to HFpEF alone. The cellular quantities of several upstream regulator correlated with indices of diastolic function (E/A ratio, Tau and/or LVEDP). Since CDCs act via the secretion of exosomes laden with signaling cargo, it is relevant that all 7 upstream regulators could, in principle, be regulated by proteins or miRNA that are present in CDC-derived exosomes. ConclusionWe identified key cellular regulators of transcription and translation that underlie the therapeutic effects of CDCs in HFpEF, whose levels correlate quantitatively with measures of diastolic function. Among the multifarious proteomic changes associated with rat model of HFpEF which were also observed in human HFpEF samples, we propose that these regulators, and downstream effector kinases, be prioritized for further dedicated mechanistic dissection.

molecular biology↗