Molecular & Cellular Proteomics最新文献

Juvenile-type granulosa cell tumors (JGCTs) manifest during the prepubertal period as precocious pseudo-puberty and/or dysmenorrhea. We have previously identified pathogenic variants in AKT1 in JGCTs. This study aims to understand how these variants affect cellular function at the phenotypic and molecular levels using a Drosophila model. Transgenic Drosophila models expressing WT AKT1 and four pathogenic variants were created under the control of tissue-specific promoters. Phenotypic effects were studied by assessing Drosophila wings for cell division and growth using wing surface and trichome density and ovarian follicular cells were examined for subcellular localization and morphology. Molecular analyses included mass spectrometry to identify differentially expressed proteins (DEPs) and phospho-peptides, along with RNA-Seq to characterize transcriptomic changes. Wings expressing mutated AKT1 showed increased surface area and reduced trichome density, indicating larger cells. In ovarian follicular cells, WT AKT1 localized primarily to the cytoplasm, while mutated AKT1 variants were associated with the plasma membrane, leading to morphological abnormalities and increased cell size. Mass spectrometry revealed numerous DEPs and phospho-peptides, highlighting changes in pathways such as glycolysis and Rho GTPase signaling. Transcriptomics demonstrated a clear gain of function for mutated AKT1 in activating a subset of genes. However, several genes upregulated by WT AKT1 were less effectively activated by the mutants, indicating a potential loss-of-function in transcriptional regulation for this subset, revealing an unexpected mechanistic complexity. Network analysis of interactions involving DEPs, phosphorylated proteins, and transcription factors suggests these elements mediate the observed proteomic and transcriptional alterations. Taken together, the results underscore the utility of Drosophila models in unraveling the biological relevance of AKT1 pathogenic variants in cancer.

Glycopeptide tandem mass spectra typically contain numerous glycan-specific fragments that can inform several features of glycan modifications, including glycan class, composition, and structure. While these fragment ions are often straightforward to observe by eye, few tools exist to systemically explore these common glycopeptide spectral features or explore their relationships with each other. Instead, most studies rely on manual inspection to understand glycan-informative ion content in their data, or they are restricted to evaluating the presence of these ions only in the small fraction of spectra that are identified by glycopeptide search algorithms. Here we introduce GlyCounter as a freely available, open-source tool to rapidly extract oxonium, Y-type, and custom ion information from raw data files. We highlight GlyCounter's utility by evaluating glycan-specific fragments in a diverse selection of publicly available datasets to demonstrate how others in the field can make immediate use of this software. In several cases, we show how conclusions drawn in these publications are evident simply through GlyCounter's extracted ion information without requiring database searches or experiment-specific programs. Although one of our goals is to decouple spectral evaluation from glycopeptide identification, we also show that evaluating oxonium ion content with GlyCounter can supplement a database search as valuable spectral evidence to validate conclusions. In all, we present GlyCounter as a user-friendly platform that can be easily incorporated into most glycoproteomic workflows to refine sample preparation, data acquisition, and post-acquisition identification methods through straightforward evaluation of the glycan content of glycoproteomic data. Software and instructions are available at https://github.com/riley-research/GlyCounter.

Hydroxychloroquine (HCQ) and chloroquine have been utilized as antimalarial drugs for decades. Recently, these compounds were reported to inhibit various viruses utilizing the endosomal entry pathway. However, their direct molecular targets in host cells remain elusive. In this study, we developed a clickable photo-crosslinking probe in combination with proteomic approaches to identified cathepsin L (CTSL) as the binding target of HCQ. Extensive biochemical and in silico analyses were conducted to validate the HCQ-CTSL interactions. HCQ significantly inhibited the protease activity of CTSL and suppressed CTSL-dependent coronavirus entry in cells that support endosomal entry pathway. These findings not only reveal the underlying mechanism of how HCQ inhibits endosomal viral entry but also guide the rational use of HCQ against other emerging infectious agents.

Evolution of multicellular life forms has involved adaptation of organs that consist of multiple cell types, each with unique functional properties that as a collection, achieve complex organ function. Since each cell type is adapted to deliver specific functionality within the context of an organ, knowledge on functional landscapes occupied by individual cell types could improve comprehension of organ function at the molecular level. In kidney, podocytes and tubules are 2 cell types of the nephron, each with vastly different functional roles. Podocytes envelop the blood vessels in the glomerulus and act as filters while tubules, located downstream of the glomerulus, are responsible for reabsorption of important nutrients. Mitochondria hold a critical and well-studied role in tubules due to the high energetic requirements required to fulfill their function. In podocytes however, questions remain regarding the relevance of mitochondrial function in both normal physiology and pathology. Quantitative cross-linking mass spectrometry and proteomics together with a transgenic mitochondrial tagging strategy were used to investigate kidney cell-type specificity of mitochondria. These efforts revealed that despite similarities of podocyte and tubule mitochondrial proteomes, each contain unique features corresponding to known distinct functional roles. These include increased demand for energy production through the tricarboxylic acid cycle in tubules and increased detoxification demand in podocytes. Moreover, tubule and podocyte mitochondrial interactome differences revealed additional cell-type specific functional insights with alterations in betaine metabolism, lysine degradation, and other pathways not regulated through proteome abundance levels. Most importantly, these efforts illustrate that cell specific mitochondrial interactome differences within an organ can now be visualized. Therefore, this approach can generally be used to map cell-specific mitochondrial changes in disease, aging or even with therapy to better understand the roles and contributions of each cell type in normal physiology and pathology within an organ in ways not previously possible.

Pathogenic variants in the CACNA1F gene are linked to congenital stationary night blindness type 2 though their specific molecular effects remain elusive. This study examines the retinal impact of two variants: a truncation (RX) and a gain-of-function (IT) to explore variant-specific retinal proteome changes. Electroretinography showed that RX primarily affects rod pathways, while IT disrupts both rod and cone signaling, consistent with morphological findings. Comprehensive quantitative proteomic analysis using mass spectrometry identified approximately 4000 proteins across wild-type control and mutant retinas, including also low-abundant membrane proteins. IT retinas exhibited widespread proteomic remodeling suggesting broad cellular responses and also compensatory molecular adaptations. In contrast, RX retinas displayed a more restricted profile. Similar to IT retinas, we found reduced Cav1.4 protein levels but without transcriptional downregulation in RX, alongside selective changes in synaptic proteins such as Erc1, Lrfn2, vGlut1, and Rab3a. These findings suggest selective molecular changes in synaptic organization and calcium-related pathways in RX retinas, offering insights into the mechanisms of Cav1.4 dysfunction in retinal disease. Deep proteomic analysis demonstrates how retinal cells reorganize their molecular architecture in response to calcium channel defects and highlights the utility of comprehensive proteomics to characterize adaptive cellular responses to genetic perturbations in retinal synaptic organization.

Total N-glycome in blood serum or plasma provides information about all serum/plasma protein enzymatic glycosylation, a tightly regulated cotranslational and post-translational modification. Total plasma/serum N-glycome has shown specific patterns (signatures) in patients with high-mortality pathologies, such as cancer and cardiovascular diseases; thus, we explored the capacity of total serum N-glycome to predict mortality in a general adult population. This prospective cohort study was performed in a municipality in Spain including a random sample of 1516 adults. Participants were profiled for total serum N-glycome at baseline. Serum enzymatic N-glycan release was performed on a robotic platform followed by hydrophilic interaction chromatography-ultraperformance liquid chromatography glycan separation. The computerized medical records were checked at a median follow-up of 7.52 years to collect the date and cause of all deaths. N-glycan groups from total serum were used to develop mortality prediction models. Total serum N-glycome peak (GP) 16, mainly composed of A2[3]BG1S[3]1, predisposed to all-cause mortality; GP 22, mainly composed of FA2G2S[6]1, protected from all-cause mortality. The balance between them predicted all-cause mortality incidence over time (area under the curve [AUC], 0.810 [0.773-0.847]). Similar results were obtained for cancer mortality, with GPs 16, 17, 22, and 23 (AUC, 0.786 [0.728-0.843]); and for cardiovascular mortality, with GPs 7 and 9 (AUC, 0.747 [0.645-0.850]). Their predictive powers had an independent and additive effect on classical prediction factors. The balances between specific GPs are independent predictors of all-cause, cancer, and cardiovascular mortality and could contribute significantly to improving prognostic tools.

The overarching symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is post-exertional malaise (PEM), an exacerbation of symptoms following physical or mental exertion. To investigate the molecular underpinnings of PEM, we performed longitudinal plasma proteomics using the Somascan 7K aptamer-based assay to monitor 6361 unique plasma proteins in 132 individuals (96 females and 36 males) subjected to two maximal cardiopulmonary exercise tests separated by a 24-h recovery period. The cohort included 79 ME/CFS cases compared to 53 age- and BMI-matched sedentary controls, allowing us to distinguish disease-specific molecular alterations from those due to physical deconditioning. Longitudinal profiling revealed widespread proteomic changes following exertion, with the most pronounced alterations observed in ME/CFS participants during the recovery phase, coinciding with the onset of PEM. Compared to controls, patients with ME/CFS showed persistent dysregulation of immune, metabolic, and neuromuscular pathways. Key findings included suppression of T and B cell signaling, downregulation of IL-17 and cell-cell communication pathways, and upregulation of glycolysis/gluconeogenesis, suggestive of mitochondrial stress and impaired immune recovery from exercise. Proteomic associations with physiological performance (VO₂max, anaerobic threshold) revealed disruptions between protein abundance and exercise capacity in ME/CFS versus controls. Correlations with symptom severity linked changes in immune-related proteins and ME/CFS symptoms, including muscle pain, recurrent sore throat, and lymph node tenderness. Sex-stratified analyses revealed distinct molecular responses between females and males, emphasizing the importance of considering sex as a biological variable in ME/CFS research. Finally, our analysis of sedentary controls contributes new data on molecular responses to acute exertion in a predominantly female sedentary cohort, a population historically underrepresented in exercise physiology studies. Together, these findings underscore the value of dynamic, proteomic profiling over time for characterizing maladaptive responses to exertion in ME/CFS and provide a foundation for deeper mechanistic investigation into PEM.

Selecting the optimal biofluid for accurate biomarker assessment is vital to an informative clinical assay. However, in the initial stages of candidate biomarker discovery, the biologically appropriate biofluid might be unclear. To resolve this dilemma, we demonstrate a mass spectrometry-based workflow where paired urine, plasma, and serum samples are processed in parallel, creating biofluid-specific peptide libraries. These libraries are then harmonized to monitor consistent peptides and transitions, enabling cross-fluid normalization and quantitative comparisons. We also present a reference dataset, "CATalog," to aid in determining which biofluid to pursue based on protein relative abundance in healthy feline urine, plasma, and serum. Using this workflow and database, we explore the interchangeability of blood biofluid proteins compared to urinary proteins relating to sample processing, relative protein quantification, and clinical application. Our results suggest that when processed correctly, urine could sometimes represent blood biofluid proteins without requiring venipuncture or sample depletion of highly abundant proteins.

Crosslinking mass spectrometry (MS) is a powerful approach for probing protein structures. However, most widely used crosslinkers rely on N-hydroxysuccinimide (NHS) esters, restricting reactivity primarily to lysine residues and protein N-termini, and rendering them incompatible with many amine-containing buffers (e.g., Tris) and key biochemical cofactors (e.g., ATP). To address these limitations, we introduce two novel vinyl-sulfone-based crosslinkers. Alkyne-BVSC is an enrichable, homobifunctional crosslinker featuring an acid-cleavable alkyne handle for downstream peptide enrichment. VSD is a heterobifunctional crosslinker combining a vinyl sulfone with a diazirine moiety for UV-activated photo-crosslinking. Both reagents are synthetically accessible from inexpensive precursors and retain reactivity in amine-rich biochemical environments. We show that vinyl sulfones react with cysteine, histidine, and lysine residues, thereby expanding crosslinkable residues beyond those accessible to NHS-esters. Moreover, we develop a stub-based post-search filtering strategy that leverages the MS-cleavable nature of vinyl sulfone linkages to improve crosslink identification sensitivity. Together, these advances establish vinyl-sulfone-based crosslinkers as versatile and complementary tools for structural proteomics.

Cell surface proteins (CSPs) regulate key cellular functions and represent valuable targets for diagnostics and therapeutics. Despite advances in proteomic workflows, CSP analysis from cryopreserved or low-input clinical samples remains limited by technical constraints, including reduced membrane integrity, inefficient labeling, and high background. To address these challenges, we optimized and benchmarked two complementary surface enrichment strategies compatible with low-input applications (fewer than 1 million cells) and real-world sample types, including viably cryopreserved cells and dissociated solid tissues. We systematically compared oxidation-based N-glycopeptide capture and wheat germ agglutinin-horseradish peroxidase-mediated proximity labeling across a range of input amounts using both solid tumor (A549) and hematologic cancer (KMS-12-BM) cell lines. The N-glycopeptide method yielded superior specificity in low-input contexts, while the wheat germ agglutinin-horseradish peroxidase method captured complementary CSP subsets. Together, the methods identified more than 700 CSPs, with approximately 175 unique identifications per protocol. Both workflows detected dynamic epidermal growth factor receptor internalization following epidermal growth factor stimulation and maintained high reproducibility (Pearson correlation greater than 0.9) between fresh and cryopreserved preparations. To extend these findings to tissue-derived samples, we optimized dissociation protocols for healthy endometrium and applied the N-glycopeptide method to cryopreserved dissociated endometrium from three healthy donors. Enzymatic dissociation enabled accurate CSP profiling from less than 1 to 2 million cells. This study provides a systematic comparison of two leading surface proteomics approaches, validates their performance on cryopreserved and low-input specimens, and demonstrates applicability to clinically relevant tissues. Our optimized workflows enable robust surfaceome characterization in translational settings where sample quantity and preservation methods are often limiting, opening new avenues for biomarker discovery and patient stratification.