Biology subjects

Rodin, S.

Publications and source records attributed to Rodin, S..

6 recordsLinked to original sources

bioRxiv2025

Single-cell proteomics of pancreatic islet cells reveals type 1 diabetes and donor-specific features

Type 1 diabetes mellitus (T1DM) is the most common severe chronic disease in children and adolescents and requires life-long exogenous insulin treatment. Investigating key differences between the pancreas of healthy and T1DM patients is key to help uncover new treatments. Here we performed a single-cell proteomic (SCP) analysis of purified pancreatic islets from several healthy and T1DM donors at a depth of > 4000 protein quantified. We employed tools designed for single-cell mRNA sequencing (scRNAseq) for cell type annotation highlighting discrepancies between mRNA and protein markers. Subsequently, we defined a subset of markers useful for future proteomics analyses of pancreatic islets. This marker list was employed to refine the annotations of our combined dataset, which we exploited with a machine learning approach to successfully transfer annotations into a new islet dataset. Lastly, we showcase the applicability and value of SCP in a clinical context by providing novel insights into cell type-specific differences between healthy and T1DM islets as well as between healthy islets of different donors. Our study is the first SCP analysis of pancreatic islets from multiple donors and T1DM islets, performed at this level of analytical depth. This provides methodological and biological insight as well as a reference dataset for future SCP studies and for any researcher interested in T1DM and in pancreatic islet physiology.

biochemistry↗

bioRxiv2025

Advancing clinical outcome predictions via incorporating pharmacokinetic simulations into in vitro testing - a colorectal cancer example

The development of in vitro assays that can predict clinical outcomes is highly desirable for drug development and personalized medicine. However, conventional in vitro methods often fail to replicate physiological drug pharmacokinetics, posing a challenge to their clinical translation. To address this issue, we adjusted incubation times and concentrations of standard-of-care drugs in the in vitro chemosensitivity assay to reflect those encountered by colorectal cancer patients. Then, for first time, we mimicked the relevant drug exposure of mFOLFOX-6, CapOx and FOLFIRI protocols to predict clinical outcomes. Our pharmacokinetic-based testing on primary colorectal cancer cells accurately predicted responders and non-responders among a cohort of patients (N=6). Classical testing methods such as IC50 and GI50 did not reveal any clinically meaningful results. Furthermore, we demonstrated that even subtle changes in drug incubation times could lead to significant variations in the classification of cells as sensitive and resistant, which is not related to mechanisms of action according to categorical clustering. Finally, our pharmacokinetic-based test results were consistent with the historical clinical data on similarities of mFOLFOX-6 and CapOx schemes. Our results contribute to the growing body of evidence that pharmacokinetic-based in vitro testing could bridge the gap between laboratory research and clinical practice. Integration of pharmacokinetic dynamics into in vitro tests could have a significant potential in enhancing drug development and refining personalized treatment strategies.

pharmacology and toxicology↗

bioRxiv2024

Global analysis of protein turnover dynamics in single cells

Even with recent improvements in sample preparation and instrumentation, single-cell proteomics (SCP) analyses mostly measure protein abundances, making the field unidimensional. In this study, we employ a pulsed stable isotope labeling by amino acids in cell culture (SILAC) approach to simultaneously evaluate protein abundance and turnover in single cells (SC-pSILAC). Using state-of-the-art SCP workflow, we demonstrated that two SILAC labels are detectable from [~]4000 proteins in single HeLa cells recapitulating known biology. We investigated drug effects on global and specific protein turnover in single cells and performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSC) encompassing six sampling times over two months and analyzed >1000 cells. Abundance measurements highlighted cell-specific markers of stem cells and various organ-specific cell types. Protein turnover dynamics highlighted differentiation-specific co-regulation of core members of protein complexes with core histone turnover discriminating dividing and non-dividing cells with potential in stem cell and cancer research. Our study represents the most comprehensive SCP analysis to date, offering new insights into cellular diversity and pioneering functional measurements beyond protein abundance. This method distinguishes SCP from other single-cell omics approaches and enhances its scientific relevance in biological research in a multidimensional manner.

systems biology↗

bioRxiv2023

SpotLight proteomics identifies variable sequences of blood antibodies specific against deamidated human serum albumin

Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in blood of patients with Alzheimers disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.

immunology↗

bioRxiv2022

Testing the link between isoaspartate and Alzheimer's disease etiology

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts the secondary and higher order structures of proteins, damaging their functions and making them prone to aggregation. An association has been suggested between isoAsp and Alzheimers Disease (AD). Here we strengthened the link between isoAsp and AD by novel approaches to isoAsp analysis in blood human serum albumin (HSA), the most abundant blood protein, a major carrier of amyloid beta (A{beta}) peptide and phosphorylated tau (pTau) protein in blood and a key participant in their clearance pathway. We discovered a reduced amount of anti-isoAsp antibodies (P < .0001), an elevated isoAsp level in HSA (P < .001), more HSA aggregates (P < .0001) and increased levels of free A{beta} (P < .01) in AD blood compared to healthy controls. We also found that deamidation significantly reduces HSA capacity to bind with A{beta} and pTau (P < .05). These findings support the presence in AD of a bottleneck in clearance of A{beta} and pTau leading to their increased concentrations in brain and facilitating their aggregations there. RESEARCH IN CONTEXTO_LISystematic review: We reviewed the evidence that associates isoaspartate (isoAsp) residue in blood proteins with the etiology of Alzheimers disease (AD). However, the link between isoAsp in blood and aggregation of amyloid beta (A{beta}) peptide and phosphorylated tau (pTau) protein in brain remained unclear. C_LIO_LIInterpretation: For the first time we demonstrate that isoAsp-containing human serum albumin (HSA) forms aggregates with reduced binding capacity toward A{beta} peptide and pTau protein. Using a novel ELISA, we discovered in AD blood elevated levels of isoAsp in HSA, together with reduced endogenous anti-isoAsp antibody levels, suggesting hampered A{beta} and pTau clearance in AD. C_LIO_LIFuture directions: As degradation of the innate anti-isoAsp defenses may take years to develop, investigation of the isoAsp role in early stages of AD is warranted. And enrollment of different neurodegenerative disease cohorts will illustrate if isoAsp is AD-specific or universal to diseases related to aging. C_LI O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

neuroscience↗

bioRxiv2018

System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA)

Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

biochemistry↗