4 research outputs found
Quantitative proteomic analysis of pancreatic cyst fluid proteins associated with malignancy in intraductal papillary mucinous neoplasms
Abstract Background The application of advanced imaging technologies for identifying pancreatic cysts has become widespread. However, accurately differentiating between low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasive intraductal papillary mucinous neoplasms (IPMNs) remains a diagnostic challenge with current biomarkers, necessitating the development of novel biomarkers that can distinguish IPMN malignancy. Methods Cyst fluid samples were collected from nine IPMN patients (3 LGD, 3 HGD, and 3 invasive IPMN) during their pancreatectomies. An integrated proteomics approach that combines filter-aided sample preparation, stage tip-based high-pH fractionation, and high-resolution MS was applied to acquire in-depth proteomic data of pancreatic cyst fluid and discover marker candidates for IPMN malignancy. Biological processes of differentially expressed proteins that are related to pancreatic cysts and aggressive malignancy were analyzed using bioinformatics tools such as gene ontology analysis and Ingenuity pathway analysis. In order to confirm the validity of the marker candidates, 19 cyst fluid samples were analyzed by western blot. Results A dataset of 2992 proteins was constructed from pancreatic cyst fluid samples. A subsequent analysis found 2963 identified proteins in individual samples, 2837 of which were quantifiable. Differentially expressed proteins between histological grades of IPMN were associated with pancreatic diseases and malignancy according to ingenuity pathway analysis. Eighteen biomarker candidates that were differentially expressed across IPMN histological grades were discovered—7 DEPs that were upregulated and 11 that were downregulated in more malignant grades. HOOK1 and PTPN6 were validated by western blot in an independent cohort, the results of which were consistent with our proteomic data. Conclusions This study demonstrates that novel biomarker candidates for IPMN malignancy can be discovered through proteomic analysis of pancreatic cyst fluid
Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation
The
development of systematic proteomic quantification techniques
in systems biology research has enabled one to perform an in-depth
analysis of cellular systems. We have developed a systematic proteomic
approach that encompasses the spectrum from global to targeted analysis
on a single platform. We have applied this technique to an activated
microglia cell system to examine changes in the intracellular and
extracellular proteomes. Microglia become activated when their homeostatic
microenvironment is disrupted. There are varying degrees of microglial
activation, and we chose to focus on the proinflammatory reactive
state that is induced by exposure to such stimuli as lipopolysaccharide
(LPS) and interferon-gamma (IFN-γ). Using an improved shotgun
proteomics approach, we identified 5497 proteins in the whole-cell
proteome and 4938 proteins in the secretome that were associated with
the activation of BV2 mouse microglia by LPS or IFN-γ. Of the
differentially expressed proteins in stimulated microglia, we classified
pathways that were related to immune-inflammatory responses and metabolism.
Our label-free parallel reaction monitoring (PRM) approach made it
possible to comprehensively measure the hyper-multiplex quantitative
value of each protein by high-resolution mass spectrometry. Over 450
peptides that corresponded to pathway proteins and direct or indirect
interactors via the STRING database were quantified by label-free
PRM in a single run. Moreover, we performed a longitudinal quantification
of secreted proteins during microglial activation, in which neurotoxic
molecules that mediate neuronal cell loss in the brain are released.
These data suggest that latent pathways that are associated with neurodegenerative
diseases can be discovered by constructing and analyzing a pathway
network model of proteins. Furthermore, this systematic quantification
platform has tremendous potential for applications in large-scale
targeted analyses. The proteomics data for discovery and label-free
PRM analysis have been deposited to the ProteomeXchange Consortium
with identifiers and , respectively
Altered secretome by diesel exhaust particles and lipopolysaccharide in primary human nasal epithelium
© 2022 The AuthorsBackground: Airway epithelial cells can actively participate in the defense against environmental pathogens to elicit local or systemic inflammation. Diesel exhaust particles (DEP), a main component of urban air pollution with particulate matter, are associated with the occurrence of acute and chronic upper airway inflammatory diseases. Objectives: We sought to investigate the effect of DEP alone or in combination with lipopolysaccharide on the secretome in the primary human nasal epithelium (PHNE) and to find potential biomarkers to relate DEP exposure to upper airway inflammatory diseases. Methods: PHNE was cultured at an air–liquid interface to create a differentiated in vivo–like model. Secreted proteins (secretome) on the bottom media of the PHNE were analyzed by mass spectrometry–based label-free quantitative proteomics and ELISA. Results: Considerably more differentially expressed secreted proteins were identified in response to DEP plus lipopolysaccharide than to DEP alone. Some canonical pathways related to inflammation and cancer such as the p53, β-catenin, and extracellular signal-regulated kinase 1/2 pathways were involved. Among differentially expressed secreted proteins, leukemia inhibitory factor was also detected at a high level in the middle ear effusions of otitis media patients, and the leukemia inhibitory factor level was significantly correlated with daily mean mass concentrations of atmospheric particulate matter averaged over 8 days before sample collection. Conclusions: Apical stimulation with DEP and lipopolysaccharide can significantly alter the basal secretome in PHNE, and this alteration can be reflected by surrounding inflammation with effusion of fluids in vivo such as middle ear effusions in otitis media patients.N