Vladislav A. Petyuk

With a sub-3-µm cell and a 27-Mbp diploid genome, the small but mighty Picochlorum celeri doubles within three hours and exceeds 30 g m ⁻² d ⁻¹ outdoors with CO ₂ supplied. In climate-simulating photobioreactors, productivity declined from 39 g m ⁻² d ⁻¹ under 2.25% CO ₂ to near zero in air and resumed within hours of CO ₂ restoration with photosystem II photochemistry largely retained. We profiled the transcriptome, proteome, phosphoproteome, ubiquitinome, acetylome, metabolome and lipidome, including the first site-resolved green-algal ubiquitinome. In air, the Rubisco large subunit tripled to 7.4% of protein while its two small-subunit isoforms, 70% identical, exchanged near-reciprocally, a novel CO ₂ -dependent switch that we propose retunes Rubisco to low CO ₂ . Photorespiratory enzymes increased in abundance whereas proteins involved in external nitrogen assimilation decreased, consistent with increased reliance on internal nitrogen recycling as carbon fixation became limiting. Transcript and protein responses were nearly uncoupled, with cytosolic ribosomal proteins declining despite increased transcripts and plastid ribosomal proteins spared. Phosphorylation was extensively remodeled, as were the lysine modifications ubiquitination, acetylation and CO ₂ -dependent carbamylation, where a CO ₂ -competition assay identified 86 bicarbonate-protected lysines, including RbcL K252. CO ₂ limitation therefore imposes a reversible growth arrest that reduces cytosolic translational investment while preserving photosynthetic capacity for rapid recovery.

Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field. However, current SCP approaches generally employ sub-μL to 1 μL processing volumes for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to compensate for evaporation, which limits their broad accessibility and analytical robustness. Here, we report a robust and convenient SCP method termed iSOP (improved surfactant-assisted one-pot processing) for the processing of single cells at a low μL processing volume using a 384-well plate with tight sealing to avoid sample drying loss. After systematic optimization, 3 μL was selected for iSOP as the processing volume, with a mixture of trypsin and Lys-C enzymes (2 ng of each enzyme) in terms of robustness, sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify ∼1200–1800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines enabled reliable identification of an average of ∼1700 and ∼2050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective, quantitative SCP analysis.

Psychological traits reflecting neuroticism, depressive symptoms, loneliness, and purpose in life are risk factors of AD dementia; however, the underlying biological mechanisms remain largely unknown. Using multi-omic data from the dorsolateral prefrontal cortex of 822 decedents in the Religious Orders Study and Rush Memory and Aging Project, we utilized a previously derived multi-omic brain molecular pseudotime representing molecular distance from no cognitive impairment (NCI) to AD dementia, and three distinct multi-omic brain molecular subtypes of AD dementia. We first confirmed generalizability of pseudotime and subtypes in two independent samples. We then annotated the subtypes, and explored whether they differed by neuropathologic burden, brain morphology or genetic risk, and found that while these indices differentiated all subtypes from NCI they did not differentiate amongst them. Finally, we tested for differential associations between the psychological traits and the subtypes, adjusting first for age, sex, education, and time to death, and then additionally for 9 common AD and Related Dementias pathologies. We found that in fully adjusted models, neuroticism, loneliness and purpose in life remained differentially associated with some AD subtypes relative to NCI. Our results are consistent with a two-stage model in which (i) upstream genetic risk influences overall disease liability, while (ii) intermediary psychological predispositions align more directly with subtype differentiation capturing AD-related heterogeneity not explained by neuropathology or brain atrophy. These results indicate that psychological risk factors may be associated with AD dementia via multi-omic molecular pathways, predominantly informed by metabolomic dysregulation, capturing heterogeneity not explained by neuropathology.

Early detection determines melanoma outcomes, yet current screening relies on visual inspection that misses molecular changes preceding clinical diagnosis. To identify serum protein signatures of melanoma development, we leveraged the Department of Defense Serum Repository, analyzing 390 longitudinal serum samples from 73 melanoma cases and matched controls across four timepoints (4 and 2 years prior, diagnosis, and 2 years post-diagnosis) using data-independent acquisition mass spectrometry with Seer Proteograph nanoparticle enrichment. We quantified 3,364 proteins and applied machine-learning strategies combining cross-sectional case-control comparisons with longitudinal tracking of within-individual changes. Cross-sectional analysis at diagnosis achieved AUC 0.823 (95% CI: 0.723-0.923), identifying eight consensus features selected in >50% of cross-validation folds: PRSS1, GSK3B, FAM20C, CES1, CCL14, EPHA10, LMAN2, and ITIH1. These signatures, spanning proteases, immune activation, and extracellular matrix remodeling, demonstrate proof-of-concept for serum-based melanoma detection and provide candidate biomarkers for validation.

Single‐cell omics tools provide unique insights into heterogeneous cell populations and their responses to stimuli. For example, single‐cell RNA sequencing has identified several transcriptionally distinct populations of microglia, which are resident immune cells of the central nervous system (CNS) that are responsive to CNS injury, infection, and neurodegeneration. To date, single‐cell studies of microglia have focused on RNA‐sequencing or cytometry by time of flight (CyTOF), which provide indirect readouts of protein abundance or quantification of a limited number of targets. Herein, we present a workflow based on FACS‐assisted isolation, cryopreservation, and nanodroplet‐based processing for single‐cell mass spectrometry proteomics analysis of the postmortem human brain cortex‐derived microglia. From a single microglial cell, 1039 proteins could be identified on average. As a proof‐of‐principle, we applied single‐cell proteomics for exploring the heterogeneity of brain microglia at the cellular level. This pilot proteomics data partially recapitulates the prior microglia subtypes. Specifically, we determined that mitochondrial proteins, in particular members of NADH dehydrogenase (Complex I), cytochrome b‐c1 (Complex III), cytochrome c oxidase (Complex IV), F1‐ATPase (Complex V), and Na+/K+‐ATPase complex, drive variation across microglia. This pipeline offers the potential for identifying functionally and analytically relevant protein targets for microglia in Alzheimer's disease and other neurological disorders. Significance of the Study: Microglia are a key brain cell type that may contribute to pathogenesis in neurodegenerative disease. Transcriptomic profiling of microglia from the central nervous system of humans and animal models has identified several subtypes of microglia, and complementary proteomic profiling of microglia is likely to provide functionally and therapeutically relevant targets. Single‐cell proteomics studies of human‐derived microglia are lacking. This work describes a label‐free, single‐cell proteomics approach for microglia isolated by fluorescence‐activated cell sorting from a human donor that yields comparable numbers of identifications in comparison to prior single‐cell RNA sequencing studies of microglia. This approach holds promise for enabling large‐scale proteomics‐based subtyping of microglia and studying their roles in neurodegenerative diseases.

β-Amyloid (Aβ) deposition is a hallmark of both Alzheimer’s disease and cerebral amyloid angiopathy. Whilst insoluble Aβ aggregates have been extensively studied, the role of soluble Aβ monomers in vascular amyloid pathology—and their association with cognitive decline—remains unclear in plaque-free brains. This study examined whether soluble cortical Aβ species are associated with cognitive outcomes and amyloid-related pathologies, including cerebral amyloid angiopathy, in the absence of parenchymal Aβ deposition. We examined post-mortem cortical tissue from nearly 200 individuals without parenchymal Aβ deposition, drawn from two longitudinal community-based cohorts. Soluble Aβ37, Aβ40 and Aβ42 were quantified by immunoassays, and total Aβ levels were measured using selected reaction monitoring proteomics. Associations with semiquantitative cerebral amyloid angiopathy burden and longitudinal cognitive trajectories were assessed using regression models adjusting for age, sex and education. Higher levels of soluble Aβ—particularly longer species such as Aβ42, reflected by elevated Aβ42/40 and reduced Aβ37/42 ratios—were significantly associated with greater cerebral amyloid angiopathy severity. Whilst immunoassay based total Aβ and Aβ ratio measures showed limited associations with cognitive outcomes, total Aβ levels quantified by selected reaction monitoring remained significantly associated with global cognitive decline. These findings support a pathogenic role for certain soluble Aβ monomers in vascular amyloid deposition. In contrast, cognitive impairment may be driven by other amyloid species such as oligomeric or extended Aβ forms. Aβ ratios may serve as specific markers for cerebral amyloid angiopathy and provide insights into early therapeutic strategies targeting vascular amyloid pathology.

Given the role of metabolism in brain health and disease, the investigation of the role of insulin (INS) and insulin-like growth factors (IGFs) as potentia

Cerebral amyloid angiopathy (CAA) is the accumulation of β-amyloid (Aβ) in the walls of small vessels in the leptomeninges and cortex and is a risk factor of intracerebral hemorrhage and dementia, but its underlying mechanism is unknown. We examined cortical proteins in relation to CAA to elucidate the molecular mechanisms underlying CAA. Data were collected from participants of 5 community-based cohorts of older adults. Participants were recruited from personal dwellings or retirement centers, were without known dementia at enrollment, and signed an Anatomic Gift Act for brain donation. At death, autopsy was performed and cortical proteins were quantified from the dorsolateral prefrontal cortex using mass spectrometry-based proteomic analysis, and pathologic indices of brain pathologies including CAA and Alzheimer disease (AD) were assessed during neuropathologic evaluations. Targeted mass spectrometry-based proteomic analysis was performed for the quantification of total Aβ protein and Aβ38 peptide. Ordinal logistic regression models were used to test the association between the proteins and CAA. A total of 887 participants were included, with a mean age at death of 89.0 (SD = 6.8) years, and 67.2% (n = 596) were women. Eighty proteins were related to CAA, of which 12 remained associated with CAA after controlling for AD pathology. However, only 4 proteins remained associated with CAA when all 12 proteins were examined in a single model: secreted modular calcium-binding protein 1 (SMOC1), secreted frizzled-related protein 1 (SFRP1), APOE, and APOE4. Examining the 4 proteins together with the 3 Aβ measures (Aβ load, total Aβ protein, and Aβ38 peptide) in a factor analysis and a structure equation model suggested 2 factors and paths: a factor including Aβ38, SFRP1, and APOE protein, which had the larger effect size in relation to CAA (standardized estimate = 0.459, SE = 0.050, p < 0.001), and a second factor including Aβ load, total Aβ protein, SMOC1, and APOE4 that had a smaller effect size in relation to CAA (standardized estimate = 0.253, SE = 0.083, p < 0.001). This study suggests 2 molecular pathways underlying CAA, with a larger effect size for the pathway including SFRP1 and APOE protein and C-terminally truncated Aβ before position 40. However, the study is an observational cross-sectional study that limits causal inference from the findings.

Background Given the role of metabolism in brain health and disease, investigating the role of insulin (INS) and insulin-like growth factors (IGFs) as potential therapeutic strategies for neurodegenerative diseases is currently underway. Yet, the signaling pathways associated with INS and IGFs in the brain remain elusive, particularly for the human brain. Unraveling these pathways is critical for harnessing their therapeutic potential in metabolism-associated brain disorders. Methods This study employed phosphoproteomics using human neuroblastoma cell line, SH-SY5Y, to unravel the signaling network of INS, IGF-1, and IGF-2. Briefly, cells were stimulated at 10 and 60-minutes with the ligands, followed by protein extraction, trypsin digestion, tandem mass tag (TMT)-labelling and phosphopeptides enrichment using an immobilized metal affinity chromatography (IMAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data was processed using R statistical software. Protein annotations were obtained from the UniprotKB database, and pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). Results Phosphoproteomics performed at 10 and 60 minutes identified 34358 phosphosites of which 3284 were significant at 10 min and 2374 at 60 min (p.adj Conclusions This study demonstrates that INS, IGF-1 and IGF-2 regulate Rho GTPase and MRTFA activation, thereby contributing to the control of actin cytoskeletal dynamics in neuronal cells. Given the role of INS and IGFs in neuronal survival and neurodegenerative conditions, elucidating the mechanisms is of critical importance, as it offers insights into disease pathogenesis and potential therapeutic targets.