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The scientific basis of intracranial aneurysm (IA) formation, its rupture and further development of cerebral vasospasm is incompletely understood. Aberrant protein expression may drive structural alterations of vasculature found in IA. Deciphering the molecular mechanisms underlying these events will lead to identification of early detection biomarkers and in turn, improved treatment outcomes. To unravel differential protein expression in three clinical subgroups of IA patients: (1) unruptured aneurysm, (2) ruptured aneurysm without vasospasm, (3) ruptured aneurysm who developed vasospasm, we performed untargeted quantitative proteomic analysis of aneurysm tissue and serum samples from three subgroups of IA patients and control subjects. Candidate molecules were then validated in a larger cohort of patients using enzyme-linked immunosorbent assay. A total of 937 and 294 proteins were identified from aneurysm tissue and serum samples, respectively. Several proteins that are known to maintain structural integrity of vasculature were found to be dysregulated in the context of aneurysm. ORM1 , a glycoprotein, was significantly upregulated in both tissue and serum samples of unruptured aneurysm patients. We employed a larger cohort of subjects ( n  = 26) and validated ORM1 as a potential biomarker for screening of unruptured aneurysms. Samples from ruptured aneurysms with vasospasm showed significant upregulation of MMP9 , a protease, compared with ruptured aneurysms without vasospasm. We validated MMP9 as a potential biomarker for vasospasm in a larger cohort ( n  = 52). This study reports the first global proteomic analysis of the entire clinical spectrum of IA. Furthermore, this study suggests ORM1 and MMP9 as potential biomarkers for unruptured aneurysm and cerebral vasospasm, respectively.

Mass spectrometry based proteomics approaches are routinely used to discover candidate biomarkers. These studies often use small number of samples to discover candidate proteins that are later validated on a large cohort of samples. Targeted proteomics has emerged as a powerful method for quantification of multiple proteins in complex biological matrix. Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) are two main methods of choice for quantifying and validating proteins across hundreds to thousands of samples. Over the years, many software tools have become available that enable the users to carry out the analysis. In this chapter, we describe selection of proteotypic peptides, sample preparation, generating a response curve, data acquisition and analysis of PRM data using Skyline software for targeted proteomics to quantify candidate markers in urine.

Shisha (water pipe) smoking is falsely believed to be a hazard-free habit and has become a major public health concern. Studies have reported shisha smoking to be associated with oral lesions, as well as carcinomas of the lung, esophagus, bladder, and pancreas. A deeper understanding of the underlying molecular mechanisms would contribute to identification of biomarkers for targeted public health screening, therapeutic innovation, and better prognosis of associated diseases. In this study, we have established an in vitro chronic cellular model of shisha-exposed oral keratinocytes to study the effect of shisha on oral cells. Normal nontransformed, immortalized oral keratinocytes were chronically exposed to shisha extract for 8 months. This resulted in significant increase in cellular proliferation and cell invasion in shisha-exposed cells compared to the parental cells. Quantitative proteomic analysis of OKF6/TERT1-Parental and OKF6/TERT1-Shisha cells resulted in the identification of 5515 proteins. Forty-three differentially expressed proteins were found to be common across all conditions. Bioinformatic analysis of the dysregulated proteins identified in the proteomic study revealed dysregulation of interferon pathway, upregulation of proteins involved in cell growth, and downregulation of immune processes. The present findings reveal that chronic exposure of normal oral keratinocytes to shisha leads to cellular transformation and dysregulation of immune response. To the best of our knowledge, this is the first report that has developed a model of oral keratinocytes chronically exposed to shisha and identified proteomic alterations associated with shisha exposure. However, further research is required to evaluate the health burden of shisha smoking.

Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly population. The current study aims to find non-invasive prognostic biomarkers in the urine specimens of the AMD patients.Methods: Peripheral blood and urine samples were collected from 23 controls and 61 AMD patients. Genomic DNA was extracted from the buffy coat of peripheral blood. Allele specific PCR was used to assay SNPs in complement factor H (CFH), complement component 3 (C3). Comparative proteomic analysis of urine samples from early AMD, choroidal neovascular membrane (CNVM), geographic atrophy (GA), and healthy controls was performed using isobaric labelling followed by mass spectrometry. Validation was performed using enzyme-linked immunosorbent assay (ELISA).Results: Comparative proteomic analysis of urine samples identified 751 proteins, of which 383 proteins were found to be differentially expressed in various groups of AMD patients. Gene ontology classification of differentially expressed proteins revealed the majority of them were involved in catalytic functions and binding activities. Pathway analysis showed cell adhesion molecule pathways (CAMs), Complement and coagulation cascades, to be significantly deregulated in AMD. Upon validation by ELISA, SERPINA-1 (Alpha1 antitrypsin), TIMP-1 (Tissue inhibitor of matrix metaloprotease-1), APOA-1 (Apolipoprotein A-1) were significantly over-expressed in AMD (n = 61) patients compared to controls (n = 23). A logistic model of APOA-1 in combination with CFH and C3 polymorphisms predicted the risk of developing AMD with 82% accuracy.Conclusion: This study gives us a preliminary data on non-invasive predictive biomarkers for AMD, which can be further validated in a large cohort and translated for diagnostic use. [ABSTRACT FROM AUTHOR]

Background: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer.Methods: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA.Results: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells.Conclusions: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer. [ABSTRACT FROM AUTHOR]

The importance of olfaction to human health and disease is often underappreciated. Olfactory dysfunction has been reported in association with a host of common complex diseases, including neurological diseases such as Alzheimer's disease and Parkinson's disease. For health, olfaction or the sense of smell is also important for most mammals, for optimal engagement with their environment. Indeed, animals have developed sophisticated olfactory systems to detect and interpret the rich information presented to them to assist in day-to-day activities such as locating food sources, differentiating food from poisons, identifying mates, promoting reproduction, avoiding predators, and averting death. In this context, the olfactory bulb is a vital component of the olfactory system receiving sensory information from the axons of the olfactory receptor neurons located in the nasal cavity and the first place that processes the olfactory information. We report in this study original observations on the human olfactory bulb proteome in healthy subjects, using a high-resolution mass spectrometry-based proteomic approach. We identified 7750 nonredundant proteins from human olfactory bulbs. Bioinformatics analysis of these proteins showed their involvement in biological processes associated with signal transduction, metabolism, transport, and olfaction. These new observations provide a crucial baseline molecular profile of the human olfactory bulb proteome, and should assist the future discovery of biomarker proteins and novel diagnostics associated with diseases characterized by olfactory dysfunction. [ABSTRACT FROM AUTHOR]

Proteomics analysis of chronic cigarette smoke exposure is a rapidly emerging postgenomics research field. While smoking is a major cause of lung cancer, functional studies using proteomics approaches could enrich our mechanistic understanding of the elusive lung cancer global molecular signaling and cigarette smoke relationship. We report in this study on a stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analysis of a human lung mucoepidermoid carcinoma cell line, H292 cells, chronically exposed to cigarette smoke. Using high resolution Orbitrap Velos mass spectrometer, we identified the hyperphosphorylation of 493 sites, which corresponds to 341 proteins and 195 hypophosphorylated sites, mapping to 142 proteins upon smoke exposure (2.0-fold change). We report differential phosphorylation of multiple kinases, including PAK6, EPHA4, LYN, mitogen-activated protein kinase, and phosphatases, including TMEM55B, PTPN14, TIGAR, among others, in response to chronic cigarette smoke exposure. Bioinformatics analysis revealed that the molecules differentially phosphorylated upon chronic exposure of cigarette smoke are associated with PI3K/AKT/mTOR and CDC42-PAK signaling pathways. These signaling networks are involved in multiple cellular processes, including cell polarity, cytoskeletal remodeling, cellular migration, protein synthesis, autophagy, and apoptosis. The present study contributes to emerging proteomics insights on cigarette smoke mediated global signaling in lung cells, which in turn may aid in development of precision medicine therapeutics and postgenomics biomarkers. [ABSTRACT FROM AUTHOR]

Chronic exposure to cigarette smoke markedly increases the risk for lung cancer. Regulation of gene expression at the post-transcriptional level by miRNAs influences a variety of cancer-related interactomes. Yet, relatively little is known on the effects of long-term cigarette smoke exposure on miRNA expression and gene regulation. NCI-H292 (H292) is a cell line sensitive to cigarette smoke with mucoepidermoid characteristics in culture. We report, in this study, original observations on long-term (12 months) cigarette smoke effects in the H292 cell line, using microarray-based miRNA expression profiling, and stable isotopic labeling with amino acids in cell culture-based quantitative proteomic analysis. We identified 112 upregulated and 147 downregulated miRNAs (by twofold) in cigarette smoke-treated H292 cells. The liquid chromatography-tandem mass spectrometry analysis identified 3,959 proteins, of which, 303 proteins were overexpressed and 112 proteins downregulated (by twofold). We observed 39 miRNA target pairs (proven targets) that were differentially expressed in response to chronic cigarette smoke exposure. Gene ontology analysis of the target proteins revealed enrichment of proteins in biological processes driving metabolism, cell communication, and nucleic acid metabolism. Pathway analysis revealed the enrichment of phagosome maturation, antigen presentation pathway, nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, and cholesterol biosynthesis pathways in cigarette smoke-exposed cells. In conclusion, this report makes an important contribution to knowledge on molecular changes in a lung cell line in response to long term cigarette smoke exposure. The findings might inform future strategies for drug target, biomarker and diagnostics innovation in lung cancer, and clinical oncology. These observations also call for further research on the extent to which continuing or stopping cigarette smoking in patients diagnosed with lung cancer translates into molecular and clinical outcomes. [ABSTRACT FROM AUTHOR]

Cigarette smoking has been associated with multiple negative effects on human skin. Long-term physiological effects of cigarette smoke are through chronic and not acute exposure. Molecular alterations due to chronic exposure to cigarette smoke remain unclear. Primary human skin keratinocytes chronically exposed to cigarette smoke condensate (CSC) showed a decreased wound-healing capacity with an increased expression of NRF2 and MMP9. Using quantitative proteomics, we identified 4728 proteins, of which 105 proteins were overexpressed (≥2-fold) and 41 proteins were downregulated (≤2-fold) in primary skin keratinocytes chronically exposed to CSC. We observed an alteration in the expression of several proteins involved in maintenance of epithelial barrier integrity, including keratin 80 (5.3 fold, p value 2.5 · 10-7), cystatin A (3.6-fold, p value 3.2 · 10-3), and periplakin (2.4-fold, p value 1.2 · 10-8). Increased expression of proteins associated with skin hydration, including caspase 14 (2.2-fold, p value 4.7 · 10-2) and filaggrin (3.6-fold, p value 5.4 · 10-7), was also observed. In addition, we report differential expression of several proteins, including adipogenesis regulatory factor (2.5-fold, p value 1.3 · 10-3) and histone H1.0 (2.5-fold, p value 6.3 · 10-3) that have not been reported earlier. Bioinformatics analyses demonstrated that proteins differentially expressed in response to CSC are largely related to oxidative stress, maintenance of skin integrity, and anti-inflammatory responses. Importantly, treatment with vitamin E, a widely used antioxidant, could partially rescue adverse effects of CSC exposure in primary skin keratinocytes. The utility of antioxidant-based new dermatological formulations in delaying or preventing skin aging and oxidative damages caused by chronic cigarette smoke exposure warrants further clinical investigations and multi-omics research. [ABSTRACT FROM AUTHOR]

Proteins present in human saliva offer an immense potential for clinical applications. However, exploring salivary proteome is technically challenged due to the presence of amylase and albumin in high abundance. In this study, we used four workflows to analyze human saliva from healthy individuals which involved depletion of abundant proteins using affinity-based separation methods followed by protein or peptide fractionation and high resolution mass spectrometry analysis. We identified a total of 1256 human salivary proteins, 292 of them being reported for the first time. All identifications were verified for any shared proteins/peptides from the salivary microbiome that may conflict with the human protein identifications. On integration of our results with the analyses reported earlier, we arrived at an updated human salivary proteome containing 3449 proteins, 808 of them have been reported as differentially expressed proteins in oral cancer tissues. The secretory nature of 598 of the 808 proteins has also been supported on the basis of the presence of signal sequence, transmembrane domain or association with exosomes. From this subset, we provide a priority list of 139 proteins along with their proteotypic peptides, which may serve as a reference for targeted investigations as secretory markers for clinical applications in oral malignancies. This article is part of a Special Issue entitled: Proteomics in India. [ABSTRACT FROM AUTHOR]