Isolation and characterization of membrane vesicles secreted by human renal cells

Most cells release membrane vesicles for various purposes including, but not limited to, intercellular communication and disposal of membrane and soluble proteins. These vesicles are secreted into urine coming from the cells lining the urinary tract and bladder epithelium. These vesicles are a promising source of biomarkers for various cardiovascular and renal diseases. This thesis pursues twofold objectives, one being the development and improvement of an isolation method for urinary membrane vesicles and the second being proteomic characterization of the content of these vesicles. These objectives are important to realise the clinical potential of these vesicles. An alternative method for removal of contaminant high-abundant proteins was developed which preserves the activity of vesicular proteins. Moreover, lipid-affinity and lectin-affinity-based novel methods to enrich membrane vesicles from minimally processed urine were evaluated and developed. More than 600 proteins were identified in urinary membrane vesicles using shotgun proteomic analysis. Post-translational modification (PTM) proteomics was carried out to identify the PTM status of vesicular proteins. Many different PTMs like glycosylation, ubiquitination and palmitoylation were assessed. Surface glycan profiles of these vesicles were elucidated using fluorophore-linked lectin assay (FLLA) employing 18 different lectins. Lectin blotting, lectin-affinity chromatography using multiple lectins and hydrazide chemistry based enrichment of glycoproteins were carried out. As a result, 108 glycoproteins were identified. Immuno-affinity chromatography was used to enrich and identify ubiquitin-conjugated proteins present in urinary membrane vesicles. A number of potential palmitoylated proteins were identified as well. Computational prediction and validation methods were applied to these protein lists. In conclusion, novel methods to isolate urinary membrane vesicles were developed. In addition, a thorough proteomic characterisation of contents of urinary membrane vesicles was achieved. This work will serve as platform for further characterization of urinary membrane vesicles

MATRIX-ISOLATION FTIR SPECTROSCOPY OF THE DEHYDRO-PYRAZINE RADICAL

Photochemistry of nitrogen-based heterocyclic radicals plays a vital role in our understanding of the fundamental chemical processes in multiple fields including combustion, and atmospheric/troposphere chemistry\footnote{Peeters, Z. et al., \textit{A\&A} \textbf{2005}, 433, 583-590.}. Photochemical studies provide insights about the mechanistic pathways and the origin of various interstellar molecules. Radical and biradical species containing heteroatoms play a significant role as intermediates in photochemical processes. The photolysis and pyrolysis studies of one such heterocycle, pyrazine have been done using various spectroscopic and computational techniques\footnote{Wilhelm, M. J. et al., \textit{J. Phys. Chem. A} \textbf{2018}, 122, 9001-9013.}. However, the photochemical investigations of the pyrazine radical have not been reported so far. In this work, we have explored the electronic structures of the radicals corresponding to all the possible diazines- pyrimidine, pyridazine, and pyrazine, at various levels of theories. Investigations for 3c-5e interactions between the two nitrogen lone pairs and the radical center have also been carried out\footnote{Saraswat, M. et al., \textit{Phys. Chem. Chem. Phys.} \textbf{2018}, 20, 4386-4395.}. This has been coupled with an experimental study; the photochemical generation and characterization of the pyrazine radical using Matrix Isolation FT-Infrared spectroscopy. Isolated pyrazine radical photochemistry in solid nitrogen and argon matrices at 4 K using strong UV irradiation leads to the formation of numerous products via ring opening and fragmentation channels, which were identified by comparing the experimental spectrum with the computationally obtained spectrum

Quantitative N-glycoproteomics reveals altered glycosylation levels of various plasma proteins in bloodstream infected patients

Bloodstream infections are associated with high morbidity and mortality with rates varying from 10–25% and higher. Appropriate and timely onset of antibiotic therapy influences the prognosis of these patients. It requires the diagnostic accuracy which is not afforded by current gold standards such as blood culture. Moreover, the time from blood sampling to blood culture results is a key determinant of reducing mortality. No established biomarkers exist which can differentiate bloodstream infections from other systemic inflammatory conditions. This calls for studies on biomarkers potential of molecular profiling of plasma as it is affected most by the molecular changes accompanying bloodstream infections. N-glycosylation is a post-translational modification which is very sensitive to changes in physiology. Here we have performed targeted quantitative N-glycoproteomics from plasma samples of patients with confirmed positive blood culture together with age and sex matched febrile controls with negative blood culture reports. Three hundred and sixty eight potential N-glycopeptides were quantified by mass spectrometry and 149 were further selected for identification. Twenty four N-glycopeptides were identified with high confidence together with elucidation of the peptide sequence, N-glycosylation site, glycan composition and proposed glycan structures. Principal component analysis, orthogonal projections to latent structures-discriminant analysis (S-Plot) and self-organizing maps clustering among other statistical methods were employed to analyze the data. These methods gave us clear separation of the two patient classes. We propose high-confidence N-glycopeptides which have the power to separate the bloodstream infections from blood culture negative febrile patients and shed light on host response during bacteremia. Data are available via ProteomeXchange with identifier PXD009048.Peer reviewe

Label-free proteomics reveals serum proteins whose levels differ between pancreatic ductal adenocarcinoma patients with short or long survival

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Changes in plasma protein levels as an early indication of a bloodstream infection

Blood culture is the primary diagnostic test performed in a suspicion of bloodstream infection to detect the presence of microorganisms and direct the treatment. However, blood culture is slow and time consuming method to detect blood stream infections or separate septic and/or bacteremic patients from others with less serious febrile disease. Plasma proteomics, despite its challenges, remains an important source for early biomarkers for systemic diseases and might show changes before direct evidence from bacteria can be obtained. We have performed a plasma proteomic analysis, simultaneously at the time of blood culture sampling from ten blood culture positive and ten blood culture negative patients, and quantified 172 proteins with two or more unique peptides. Principal components analysis, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and ROC curve analysis were performed to select protein(s) features which can classify the two groups of samples. We propose a number of candidates which qualify as potential biomarkers to select the blood culture positive cases from negative ones. Pathway analysis by two methods revealed complement activation, phagocytosis pathway and alterations in lipid metabolism as enriched pathways which are relevant for the condition. Data are available via ProteomeXchange with identifier PXD005022.Peer reviewe

Preoperative Radiotherapy Leads to Significant Differences in the Plasma Protein Profile of Rectal Cancer Patients

Introduction:Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 10% of the global cancer burden. Rectal cancer accounts for around 30% of CRC cases, and patients with resectable rectal cancer are often given preoperative radiotherapy (PRT) to reduce the rate of local recurrence. The human plasma proteome is an exceptionally complex proteome and ideal to study due to its ability to reflect the presence of diseases such as cancer and the ease of obtaining blood samples. Previous proteomic studies involving rectal cancer patients have mostly focused on the identification of proteins involved in resistance to radiotherapy.Objective:The aim of this study was to investigate the overall effects of PRT on plasma protein expression in rectal cancer patients, as there is a lack of such studies.Methods:Here, we have used mass spectrometry and subsequent statistical analyses to analyze the plasma samples of 30 rectal cancer patients according to PRT status (positive or negative) and tumor stage (II or III).Results and Conclusions:We discovered 42 proteins whose levels differed significantly between stage II and III rectal cancer patients who did or did not receive PRT. This study shows that PRT, although localized to the pelvis, leads to measurable, tumor stage-specific changes in plasma protein expression. Future studies of plasma proteins should, when relevant, take this into account and be aware of the widespread effects that PRT has on the plasma proteome.Peer reviewe

Plasma protein expression differs between colorectal cancer patients depending on primary tumor location

Colorectal cancer (CRC) includes tumors in the right colon, left colon, and rectum, although they differ significantly from each other in aspects such as prognosis and treatment. Few previous mass spectrometry-based studies have analyzed differences in protein expression depending on the tumor location. In this study, we have used mass spectrometry-based proteomics to analyze plasma samples from 83 CRC patients to study if differences in plasma protein expression can be seen depending on primary tumor location (right colon, left colon, or rectum). Differences were studied between the groups both regardless of and according to tumor stage (II or III). Large differences in plasma protein expression were seen, and we found that plasma samples from patients with rectal cancer separated from samples from patients with colon cancer when analyzed by principal component analysis and hierarchical clustering. Samples from patients with cancer in the right and left colon also tended to separate from each other. Pathway analysis discovered canonical pathways involved in lipid metabolism and inflammation to be enriched. This study will help to further define CRC as distinct entities depending on tumor location, as shown by the widespread differences in plasma protein profile and dysregulated pathways.Peer reviewe

Label-free plasma proteomics identifies haptoglobin-related protein as candidate marker of idiopathic pulmonary fibrosis and dysregulation of complement and oxidative pathways

Idiopathic pulmonary fibrosis (IPF) is a lung parenchymal disease of unknown cause usually occurring in older adults. It is a chronic and progressive condition with poor prognosis and diagnosis is largely clinical. Currently, there exist few biomarkers that can predict patient outcome or response to therapies. Together with lack of markers, the need for novel markers for the detection and monitoring of IPF, is paramount. We have performed label-free plasma proteomics of thirty six individuals, 17 of which had confirmed IPF. Proteomics data was analyzed by volcano plot, hierarchical clustering, Partial-least square discriminant analysis (PLS-DA) and Ingenuity pathway analysis. Univariate and multivariate statistical analysis overlap identified haptoglobin-related protein as a possible marker of IPF when compared to control samples (Area under the curve 0.851, ROC-analysis). LXR/RXR activation and complement activation pathways were enriched in t-test significant proteins and oxidative regulators, complement proteins and protease inhibitors were enriched in PLS-DA significant proteins. Our pilot study points towards aberrations in complement activation and oxidative damage in IPF patients and provides haptoglobin-related protein as a new candidate biomarker of IPF.Peer reviewe

Mass spectrometry-based lipidomics of oral squamous cell carcinoma tissue reveals aberrant cholesterol and glycerophospholipid metabolism - A Pilot study

Lipid metabolic reprogramming is one hallmark of cancer. Lipid metabolism is regulated by numerous enzymes, many of which are targeted by several drugs on the market. We aimed to characterize the lipid alterations in oral squamous cell carcinoma (OSCC) as a basis for understanding its lipid metabolism, thus identifying potential therapeutic targets. We compared lipid species, classes, and glycerophospholipid (GPL) fatty acid species between paired tumor tissue and healthy oral tongue mucosa samples from 10 OSCC patients using a QExactive mass spectrometer. After filtering the 1370 lipid species identified, we analyzed 349 species: 71 were significantly increased in OSCC. The GPL metabolism pathway was most represented by the lipids differing in OSCC (P = .005). Cholesterol and the GPLs phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols were most significantly increased in OSCC tissue (FC 1.8, 2.0, 2.1, and 2.3 and, P = .003, P = .005, P = .002, P = .007). In conclusion, we have demonstrated a shift in the lipid metabolism in these OSCC samples by characterizing the detailed landscape. Predominantly, cholesterol and GPL metabolism were altered, suggesting that interactions with sterol regulatory binding proteins may be involved. The FA composition changes of the GPLs suggest increased de novo lipogenesis.Peer reviewe

Label-free tissue proteomics can classify oral squamous cell carcinoma from healthy tissue in a stage-specific manner

Objectives: No prognostic or predictive biomarkers for oral squamous cell carcinoma (OSCC) exist. We aimed to discover novel proteins, altered in OSCC, to be further investigated as potential biomarkers, and to improve understanding about pathways involved in OSCC. Materials and Methods: Proteomic signatures of seven paired healthy and OSCC tissue samples were identified using ultra-definition quantitative mass spectrometry, then analysed and compared using Anova, principal component analysis, hierarchical clustering and OPLS-DA modelling. A selection of significant proteins that were also altered in the serum from a previous study (PMID: 28632724) were validated immunohistochemically on an independent cohort (n = 66) to confirm immunopositivity and location within tumour tissue. Ingenuity Pathways Analysis was employed to identify altered pathways. Results: Of 829 proteins quantified, 257 were significant and 72 were able to classify healthy vs OSCC using OPLS-DA modelling. We identified 19 proteins not previously known to be upregulated in OSCC, including prosaposin and alpha-taxilin. KIAA1217 and NDRG1 were upregulated in stage IVa compared with stage I tumours. Altered pathways included calcium signalling, cellular movement, haematological system development and function, and immune cell trafficking, and involved NF-kappa B and MAPK networks. Conclusions: We found a set of proteins reliably separating OSCC tumour from healthy tissue, and multiple proteins differing between stage I and stage IVa OSCC. These potential biomarkers can be studied and validated in larger cohorts.Peer reviewe