a-comprehensive-workflow-for-plasma-proteomics-mass-spectrometry

A Comprehensive Workflow for Plasma Proteomics by Mass Spectrometry: A Strategy for Biomarker Discovery in Systemic Human Diseases

Plasma, the cell-free component of blood, offers a minimally invasive and rich source of biomolecular information, making it ideal for investigating ...

Jubilee Mission Medical College and Research Institute

Neuromyelitis optica spectrum disorders (NMOSD) is a demyelinating autoimmune disease affecting the central nervous system causing inflammatory lesions in the optic nerves, spinal cord and other vital areas of CNS. The clinical manifestations include acute transverse myelitis with paraplegia and optic neuritis with impaired vision. In the present study we focussed on comparative expression of serum proteins between NMOSD variants and control. The study has identified a plethora of novel and unexplored acute phase proteins involved in lipid transport and myelination in perspective of NMOSD. The serum proteins obtained after albumin globulin depletion were subjected to LC-MS/MS. The commonly altered proteins in all NMOSD variants with respect to control were smaug homolog protein and serum amyloid A. Truncated breast and ovarian cancer susceptibility protein and cystic fibrosis transmembrane conductance regulator protein were specifically upregulated and can act as potential biomarkers for neuromyelitis optica with autoantibody negativity to Aquaporin - 4 (AQP-4) and myelin oligodendrocyte (MOG) immunoglobulins. In addition, the uniquely downregulated proteins such as antithrombin III and histidine rich glycoprotein in NMO/MOG autoantibody negative samples can be accounted for its dysregulated fibrinolysis associated with NMO. The differentially expressed proteins were involved in cholesterol transport, synaptic vesicle mediated transport, neurotransmission and immune regulation which are closely associated with myelin formation and protection

Identification of Novel Serum Proteins Associated with Myelination and Cholesterol Transport in Neuromyelitis Optica Spectrum Disorders by Mass Spectrometry.

Human acetylcholinesterase (hAChE) has a potential role in the management of acetylcholine, one of the neurotransmitters that modulate the overall activity of cholinergic system, AChE inhibitors have a greater impact in the therapeutics. Though the atomic structure of hAChE has been extensively studied, the precise active site geometry upon binding to different ligands are yet to be explored. In the present study, an extensive structural analysis of our recently reported hAChE-tacrine complex has carried out and revealed the presence of two prominent sub-pockets located at the vicinity of the hAChE active site. Structural bioinformatics assisted studies designed 132 putative sub-pockets focused tacrine derivatives (SPFTDs), their molecular docking, free energy estimations revealed that they are stronger than tacrine in terms of binding affinity. Our in vitro studies also supported the in silico findings, all these SPFTDs are having better potencies than tacrine. Cytotoxic nature of these SPFTDs on HepG2 and Neuro-2a cell lines, diminishes the possibilities for future in vivo studies. However, the identification of these sub pockets and the SPFTDs paved a new way to the future drug discovery especially since AChE is one of the promising and approved drug targets in treatment of AD drug discovery.

Sub-pocket-focused designing of tacrine derivatives as potential acetylcholinesterase inhibitors.

COPD is a complex respiratory disorder with high morbidity and mortality rates. Even with the current conventional diagnostic methods, including circulating inflammatory biomarkers, underdiagnosis rates in COPD remain as high as 70%. Our study was a comparative cross-sectional study that aimed to address the diagnostic challenges by identifying future biomarker candidates in COPD variants.

Exploring the novel duo of Reticulocalbin, and Sideroflexin as future biomarker candidates for Exacerbated Chronic Obstructive Pulmonary Disease.

Current diagnostic methods for JIA lack specificity, often failing to distinguish it from other childhood diseases of similar clinical presentations. The present study is a comparative cross-sectional study that identified potential biomarkers using label-free mass spectrometry and bioinformatics. Two differentially expressed proteins (DEPs), Myosin light chain 12b (Myl12b) and Mannose-binding lectin serine protease 1 (MASP1), showed increased expression in JIA patients. Receiver operating characteristic (ROC) analysis revealed strong predictive power (AUC: Myl12b = 0.757, MASP1 = 0.855), validated in a separate cohort via Western blot and ELISA. These findings suggest Myl12b and MASP1 as promising biomarkers for JIA diagnosis and treatment. Data: ProteomeXchange (PXD058863).

Myosin Light Chain 12b and MASP1 as Novel Biomarker Candidates in Active Juvenile Idiopathic Arthritis─A Combined Proteomics/Bioinformatics Approach.

Chronic Obstructive Pulmonary Disease is a major global health concern with significant morbidity and mortality, characterized by heterogeneity influenced by inflammation, oxidative stress, and autoimmunity. This study investigated the role of autoimmunity in stable and exacerbated COPD phenotypes using proteomic and immunological analyses to elucidate molecular mechanisms and identify potential biomarkers. Plasma samples from COPD phenotypes and healthy controls were analyzed using label-free mass spectrometry to identify differentially expressed proteins. Functional annotation and pathway enrichment analysis highlighted proteins linked to autoimmunity, while immunological assays assessed anti-nuclear antibody prevalence and intensity using ANA ELISA and indirect immunofluorescence. The study identified differentially expressed proteins, namely, DNA repair protein XRCC2, phosphatidyl inositol glycan-specific phospholipase D, E3 ubiquitin protein ligase SHPRH, and Protocadherin-β, implicated in autoimmune pathways. Pathway enrichment analysis of these proteins highlighted the uPAR-mediated signaling, mTOR, PI3K/Akt, ARF6, and S1P signaling pathways, known for their roles in autoimmunity. Immunological assays revealed ANA positivity in 47% of stable COPD and 36% of exacerbated COPD, among which, 80% exhibited a speckled fluorescence pattern, often associated with anti-SSA and anti-SSB antibodies. The findings highlight the potential role of autoimmunity in COPD pathogenesis, suggesting phenotype-specific immune dysregulation, providing a basis for future biomarker and therapeutic research.

Relationship between autoimmunity and COPD: an investigation based on proteomic profiling and antinuclear antibody screening.

7-Ketocholesterol (7-KC) is a biologically active oxysterol formed through the oxidation of cholesterol, predominantly under conditions of oxidative stress. It is generated both enzymatically in specific tissues such as the brain and liver, and non-enzymatically via reactive oxygen species (ROS), especially in aging tissues and heat-processed animal-derived foods. 7-KC exerts multifaceted effects on human health, extending beyond lipid metabolism to disrupt glucose and amino acid utilization, impair mitochondrial function, and provoke endoplasmic reticulum (ER) stress. These disturbances contribute to chronic inflammation and oxidative damage, playing pivotal roles in the development of various diseases, including atherosclerosis, neurodegenerative disorders, diabetes, cancer, hepatic steatosis, and ocular and gastrointestinal pathologies. Additionally, 7-KC is a marker of cholesterol oxidation in the food industry, where it signals product degradation and potential toxicity in long-stored or thermally processed animal-based foods. This review explores the biosynthesis, metabolic fate, and pathophysiological role of 7-KC, highlighting its critical role in intermediary metabolism, disease progression, and food safety. Furthermore, it outlines mitigation strategies to reduce 7-KC exposure through dietary modifications, antioxidant interventions, and advanced food processing technologies aimed at enhancing public health.

7-ketocholesterol as a critical oxysterol: Impact on human health and safety in food systems.

Inflammation is crucial for wound healing, but chronic inflammation leads to complications.  L. leaves possess anti-inflammatory properties, and this study explores the effects of the Flavonoid Fraction of  (FFCP) leaf on inflammation and wound healing. In human PBMCs, the FFCP treatment efficiently reduced the activity of COX, LOX, iNOS, NO productions, and the release of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6 into the culture media. Molecular docking revealed that glycosidic flavonoids were more potent COX-2 and 5-LOX inhibitors than aglycones. FFCP (100 μg/mL) significantly enhanced cell migration, closing the wound within 36 h. These findings suggest that FFCP possesses anti-inflammatory properties and promote fibroblast migration , highlighting the potential therapeutic application of  flavonoids in inflammation management and tissue repair.

Article	Year
Identification of Novel Serum Proteins Associated with Myelination and Cholesterol Transport in Neuromyelitis Optica Spectrum Disorders by Mass Spectrometry. Indian journal of clinical biochemistry : IJCB\| PubMed ID: 35873620	2022
Sub-pocket-focused designing of tacrine derivatives as potential acetylcholinesterase inhibitors. Computers in biology and medicine\| PubMed ID: 36841058	2023
Exploring the novel duo of Reticulocalbin, and Sideroflexin as future biomarker candidates for Exacerbated Chronic Obstructive Pulmonary Disease. Clinical proteomics\| PubMed ID: 38355435	2024
Myosin Light Chain 12b and MASP1 as Novel Biomarker Candidates in Active Juvenile Idiopathic Arthritis─A Combined Proteomics/Bioinformatics Approach. Journal of proteome research\| PubMed ID: 39932158	2025
Relationship between autoimmunity and COPD: an investigation based on proteomic profiling and antinuclear antibody screening. Journal of immunoassay & immunochemistry\| PubMed ID: 40329569	2025
7-ketocholesterol as a critical oxysterol: Impact on human health and safety in food systems. The Journal of steroid biochemistry and molecular biology\| PubMed ID: 40915548	2026
Modulation of COX-2, 5-LOX, and cytokine signalling by L. Leaf cultivar 'Red Lady' flavonoids in inflammation: and insights. Natural product research\| PubMed ID: 41022640	2025

Mathew John

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