Biology subjects

Stotland, A.

Publications and source records attributed to Stotland, A..

9 recordsLinked to original sources

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↗

bioRxiv2026

Asporin Improves Cardiac Myocyte Response to Ischemia and Reperfusion Stress

BackgroundMyocardial Infarction (MI) remains a leading cause of mortality worldwide, despite advancements in clinical therapies and interventions. MI results from prolonged ischemia, leading to hypoxia-induced damage to cardiac tissue and reperfusion-injury (R/I) that aggravates cardiomyocyte (CM) loss. One key cellular event during this process is accumulation of dysfunctional mitochondria, resulting from environmental hypoxia and subsequent oxidative stress upon reperfusion. Post-MI cardiac-remodeling involves changes in both cellular and extracellular matrix (ECM). Ubiquitin-dependent and independent autophagy are crucial for cardio protection during this phase. The ECM provides structural integrity and functions as a reservoir for signaling molecules. Asporin (ASPN), a small leucine-rich proteoglycan, plays a role in modulating cardiac-remodeling by limiting excessive fibrosis and protecting CMs from cell death. MethodsWe investigated the therapeutic potential of ASPN by using an exogenous recombinant peptide of ASPN (rASPN), testing its effects using an in-vitro ischemia-reperfusion (I/R) model simulating MI conditions. Two I/R models were developed using an immortalized human embryonic cardiac cell line to reflect the hypoxia-reperfusion (H/R) phases of MI. In the No-Reoxygenation (No-ReOx) model, cells were subjected to hypoxia for 18 hours, with or without exogenous rASPN. In the Reoxygenation (ReOx) model, cells underwent 18 hours of hypoxia, then 12 hours of reoxygenation (simulating reperfusion), with or without rASPN. ResultsProteomics revealed that ASPN modulates key pathways involved in apoptosis, non-canonical autophagy, and metabolic reprogramming. Additionally, ASPN influenced immune response pathways and significantly affected TGF-{beta} signaling, a central mediator of cardiac fibrosis and remodeling post-MI. These findings indicate that ASPN plays a multifaceted role in regulating cellular responses to hypoxia and R/I. ConclusionsOur H/R model simulates key aspects of MI and R/I. The protective role of ASPN observed in this model suggests it as a promising candidate for developing cardioprotective therapies to minimize R/I and adverse cardiac-remodeling following MI.

physiology↗

bioRxiv2025

CNPY4 is a Lipid-Binding Regulator of Sphingolipid Homeostasis

Sphingolipids function both as signaling molecules and as organizers of cell membranes, and their dysregulation has been linked to cancer, metabolic disorders, and neurodegeneration. A central node in the sphingolipid metabolism network is ceramide, which is converted into numerous derivatives, including sphingomyelin (SM). Through interactions with cholesterol, SM forms liquid-ordered microdomains that influence membrane organization and signaling. Previously, we reported that the Saposin-like (SAPLIP) protein Canopy4 (CNPY4) negatively regulates the levels of free cholesterol in the plasma membrane. Although SAPLIPs commonly regulate lipid metabolism through direct lipid interactions, CNPY4 does not bind cholesterol directly. Here, we show that CNPY4 interacts with multiple sphingolipids in vitro, including ceramide and SM, and with ceramide in cells. We also demonstrate that CNPY4 knockdown elevates SM levels at the plasma membrane and disrupts cellular localization and abundance of ceramide, suggesting that the levels and consequently the homeostatic distribution of these sphingolipids is under control of CNPY4. Although the most pronounced effect of CNPY4 loss is on the ceramide/SM conversion pathway, it additionally impacts the levels of over 150 cellular lipids and modulates neutral sphingomyelinase activity, consistent with secondary disruptions in sphingolipid homeostasis. Collectively, our findings point to CNPY4 as a sphingolipid chaperone that regulates the abundance and localization of these lipids, modulating in turn cholesterol homeostasis and cellular signaling.

biochemistry↗

bioRxiv2024

Methods for detection of cardiac glycogen-autophagy

Glycogen-autophagy ( glycophagy) is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

molecular biology↗

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↗

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↗

bioRxiv2021

Protective role of the Atg8 1 homologue Gabarapl1 in regulating cardiomyocyte glycophagy in diabetic heart disease

Diabetic heart disease is highly prevalent and characterized by diastolic dysfunction. The mechanisms of diabetic heart disease are poorly understood and no targeted therapies are available. Here we show that the diabetic myocardium (type 1 and type 2) is characterized by marked glycogen elevation and ectopic cellular localization - a paradoxical metabolic pathology given suppressed cardiomyocyte glucose uptake in diabetes. We demonstrate involvement of a glycogen-selective autophagy pathway ( glycophagy) defect in mediating this pathology. Genetically manipulated deficiency of Gabarapl1, an Atg8 autophagy homologue, induces cardiac glycogen accumulation and diastolic dysfunction. Stbd1, the Gabarapl1 cognate autophagosome partner is identified as a unique component of the early glycoproteome response to hyperglycemia in cardiac, but not skeletal muscle. Cardiac-targeted in vivo Gabarapl1 gene delivery normalizes glycogen levels, diastolic function and cardiomyocyte mechanics. These findings reveal that cardiac glycophagy is a key metabolic homeostatic process perturbed in diabetes that can be remediated by Gabarapl1 intervention.

molecular biology↗

bioRxiv2019

MitoPlex: A Targeted Multiple Reaction Monitoring Assay for Quantification of a Curated Set of Mitochondrial Proteins

Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed MitoPlex. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (218 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, MitoPlex plus metabolites indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.

molecular biology↗