Narcolepsy type 1 (NT1) is the most common type of narcolepsy known to be caused by the loss of specific neurons responsible for producing peptide neurotransmitters (orexins/hypocretins), resulting in a sleep-wake cycle disorder. It is characterized by its association with cataplexy and abnormalities in rapid eye movement. To date, no cure has been established for this life-threatening condition. Misdiagnosis of NT1 is also quite common, although it is not exceedingly rare. Therefore, successfully identifying candidate serum biomarkers for NT1 would be a head start for accurate diagnosis and development of therapeutics for this disorder. This study aims to identify such potential serum biomarkers. A depletion protocol was employed for 27 human serum samples (16 NT1 and 11 healthy controls), followed by applying LC-MS/MS bottom-up proteomics analysis, then LC-PRM-MS for validation. The comparison of the proteome profiles of the low-abundant proteins in the samples was then investigated based on age, sex, sample groups, and the presence of the Human Leukocyte Antigen (HLA) DQB1*0602 allele. The results were tracked to gene expression studies as well as system biology to identify key proteins and understand their relationship in the pathogenesis of NT1. Our results revealed 36 proteins significantly and differentially expressed. Among the impaired pathways and bioprocesses, the complement activation pathway is impaired by six of the differentially expressed proteins (DEPs). They are coded by the genes C2, CFB, C5, C1R, C1S, and MASP1, while 11 DEPs are involved in Acute Phase Response Signaling (APRS), which are coded by the genes FN1, AMBP, APOH, CFB, CP, ITIH2, C5, C2, F2, C1, and ITIH4. The combined AUCs of the downregulated and upregulated DEPs are 0.95 and 0.76, respectively. Overall, this study reveals potential serum-protein biomarkers of NT1 and explains the possible correlation between the biomarkers and pathophysiological effects, as well as important biochemical pathways involved in NT1.

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus’ active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N-glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N-glycome abundance of all variants. The N-glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc₄,Hex₅,Fuc,NeuAc (4-5-1-1) and GlcNAc₆,Hex₈,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein’s structural differences between SARS-CoV-2 mutations.

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell–cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography–mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R² = 0.9961–0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types.

Background: Restless legs syndrome (RLS) is a common sensory‐motor disorder characterized by an urge to move the legs, often with unpleasant sensations, particularly during rest. Current treatments include iron supplementation, dopamine agonists, and opioids, but new therapeutic approaches are needed. The dysfunction of the A11 nucleus, which modulates dopaminergic transmission to the spinal cord, is thought to play a role in RLS pathophysiology. Calcitonin gene‐related peptide (CGRP), which is involved in pain modulation, may interact with A11 pathways, suggesting a role in RLS.Objectives: This study aimed to assess the involvement of CGRP in RLS by determining if CGRP‐related proteins are overexpressed in RLS patients.Methods: A cross‐sectional study was conducted with 17 drug‐free RLS patients (mean age 55.8 years) and 17 age‐ and gender‐matched controls. Serum samples were analyzed using liquid chromatography‐parallel reaction monitoring‐tandem mass spectrometry (LC‐PRM‐MS/MS) to identify and quantify CGRP‐related proteins. Principal component analysis (PCA) was used to differentiate between groups.Results: PCA showed clear differentiation between RLS and control groups. Among 13 identified CGRP‐related proteins, 10 were dysregulated in RLS patients: 8 were upregulated, and 2 were downregulated, among them notable proteins such as S100A12, ADM, SRSF6, and ADM2.Conclusions: This study indicates the significant involvement of CGRP and related proteins in RLS. This suggests these proteins may play roles in various aspects of the disorder. Further research is required to validate these findings and explore their clinical implications, including development of new treatment options that specifically address CGRP pathways. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The neurological condition known as narcolepsy type 1 (NT1) is an uncommon condition marked by extreme daytime sleepiness, cataplexy, sleep paralysis, hallucinations, disrupted nocturnal sleep, and low or undetectable levels of orexin in the CSF fluid. NT1 has been hypothesized to be an immunological disorder; its treatment is currently only symptomatic, and misdiagnosis is not uncommon. This study compares the N-glycome of NT1 patients with healthy controls in search of potential glycan biomarkers using LC-MS/MS. A total of 121 candidate N-glycans were identified, 55 of which were isomeric N-glycan structures and 65 were not. Seventeen N-glycan biomarker candidates showed significant differences between the NT1 and control cohorts. All of the candidate glycan biomarkers were isomeric except HexNAc₆Hex₇Fuc₀NeuAc₁ (6701) and HexNAc₆Hex₇Fuc₁NeuAc₂ (6712). Therefore, with isomeric and nonisomeric structures, a total of 20 candidate N-glycan biomarkers are reported in this study, and interestingly, all are either sialylated or sialylated-fucosylated and upregulated in NT1 relative to the control. The distribution levels of all the identified N-glycans show that the sialylated glycan type is the most abundant in NT1 and is majorly disialylated, although the trisialylated subtype is three-fold higher in NT1 compared to the healthy control. The first isomers of HexNAc₅Hex₆Fuc₀NeuAc₃ (5603), HexNAc₆Hex₇Fuc₀NeuAc₂ (6702), and HexNAc₆Hex₇Fuc₁NeuAc₄ (6714) expressed a high level of fold changes (FC) of 1.62, 2.19, and 2.98, respectively. These results suggest a different N-glycome profile of NT1 and a relationship between sialylated glycan isomers in NT1 disease development or progression. The revelation of N-glycan expression alterations in this study may improve NT1 diagnostic methods, understanding of NT1 pathology, and the development of new targeted therapeutics.