B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma

Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas

Abstract Background Cerebrospinal fluid (CSF) is a proximal fluid which communicates closely with brain tissue, contains numerous brain-derived proteins and thus represents a promising fluid for discovery of biomarkers of central nervous system (CNS) diseases. The main purpose of this study was to generate an extensive CSF proteome and define brain-related proteins identified in CSF, suitable for development of diagnostic assays. Methods Six non-pathological CSF samples from three female and three male individuals were selected for CSF analysis. Samples were first subjected to strong cation exchange chromatography, followed by LC-MS/MS analysis. Secreted and membrane-bound proteins enriched in the brain tissues were retrieved from the Human Protein Atlas. Results In total, 2615 proteins were identified in the CSF. The number of proteins identified per individual sample ranged from 1109 to 1421, with inter-individual variability between six samples of 21 %. Based on the Human Protein Atlas, 78 brain-specific proteins found in CSF samples were proposed as a signature of brain-enriched proteins in CSF. Conclusion A combination of Human Protein Atlas database and experimental search of proteins in specific body fluid can be applied as an initial step in search for disease biomarkers specific for a particular tissue. This signature may be of significant interest for development of novel diagnostics of CNS diseases and identification of drug targets

Quantitative tandem mass-spectrometry of skin tissue reveals putative psoriatic arthritis biomarkers

Abstract Background Psoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA. Methods Skin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins. Results Forty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001). Conclusion Proteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

Abstract Background Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. Methods Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. Results The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn’s disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. Conclusions This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings

Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry

Abstract Background Synovial fluid (SF) is a dynamic reservoir for proteins originating from the synovial membrane, cartilage, and plasma, and may therefore reflect the pathophysiological conditions that give rise to arthritis. Our goal was to identify and quantify protein mediators of psoriatic arthritis (PsA) in SF. Methods Age and gender-matched pooled SF samples from 10 PsA and 10 controls [early osteoarthritis (OA)], were subjected to label-free quantitative proteomics using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the two groups. Pathway analysis and public database searches were conducted to ensure these proteins held relevance to PsA. Multiplexed selected reaction monitoring (SRM) assays were then utilized to confirm the elevated proteins in the discovery samples and in an independent set of samples from patients with PsA and controls. Results We determined that 137 proteins were differentially expressed between PsA and control SF, and 44 were upregulated. The pathways associated with these proteins were acute-phase response signalling, granulocyte adhesion and diapedesis, and production of nitric oxide and reactive oxygen species in macrophages. The expression of 12 proteins was subsequently quantified using SRM assays. Conclusions Our in-depth proteomic analysis of the PSA SF proteome identified 12 proteins which were significantly elevated in PsA SF compared to early OA SF. These proteins may be linked to the pathogenesis of PsA, as well serve as putative biomarkers and/or therapeutic targets for this disease

"Product ion monitoring" assay for prostate-specific antigen in serum using a linear ion-trap.

While numerous strategies exist for biomarker discovery, the bottleneck to product development and routine use at the clinic is in the verification phase of candidate biomarkers. The aim of this study was to establish a robust and high-throughput product ion monitoring (PIM) assay that is potentially capable of rapidly verifying candidates from discovery phase experiments. Using prostate-specific antigen (PSA), a model biomarker, and a routinely used mass spectrometer for discovery platforms, an ion trap (LTQ, Thermo), the utility of this instrument to perform PIM was explored. The proteotypic doubly charged intact peptide LSEPAELTDAVK ( m/ z 637) fragmenting to m/ z 943 (PAELTDAVK) was monitored. A limit of detection of 10 attomoles with a coefficient of variation (CV) of <20% was obtained for a purified recombinant PSA digest. Immunoextraction of endogenous PSA from serum using a monoclonal antibody on a 96-well microtiter plate, followed by PIM on the LTQ, enabled quantification of PSA down to less than 1 ng/mL with a limit of detection of 0.1 ng/mL and CVs < 20%. Mascot searching and ion ratio confirmation further supported the conclusion that the quantified moiety in serum was the PSA peptide. We conclude that this methodology could be adapted quickly and easily to other candidates, thus providing a much needed technology to bridge the gap between discovery and validation platforms

Correction to: A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

In the original version of the article [1], an error was noticed under the heading “Immunoprecipitation on protein G magnetic beads” in “Methods” section

The problematic of the culture in Ostrava (the opimon of professionals)

Záměrem této práce je nastínit obraz kultury a kulturních významů, které tvoří současné město Ostrava a přilehlý širší region, specifický genius loci a jeho vytváření, a to zmínkou o historii města, dále pomocí vybraných kulturních prvků, ale především provedeného průzkumu mezi odbornou veřejností z oblasti kultury, který má za cíl pokusit se zmapovat jaký má a může mít kultura v současné době v tak specifickém městě, jako je Ostrava, význam a v jakém stavu jsou ostravské kulturní instituce. Historii zmíním od prvního osídlení v oblasti, první písemné zmínky a následný vývoj ve středověku až po význam industriální revoluce na konci 18. a především v 19. století. V tomto významném období se pokusím nastínit hlavní migrační pohyby a nálady obyvatel a jejich motivace, zmíním demografický vývoj oblasti Ostravska se specifickým způsobem bydlení a zvyky imigrantů, kteří přicházeli do tohoto regionu. Poukáži na významné, především stavební a městotvorné počiny v období tzv. první republiky, dále v období po roce 1948, pro srovnání rovněž spíše z hlediska urbanistického, až do ukončení těžby uhlí a výroby železa na území města Ostravy. Domnívám se, že tyto elementy kultury, které měly nebo mají pro město zásadní význam, pomohou v této diplomové práci syntézou vytvořit jako mozaika určitý vjem a povědomí o městě...

Unraveling endometriosis-associated ovarian carcinomas using integrative proteomics [version 1; referees: 2 approved]

Background: To elucidate potential markers of endometriosis and endometriosis-associated endometrioid and clear cell ovarian carcinomas using mass spectrometry-based proteomics. Methods: A total of 21 fresh, frozen tissues from patients diagnosed with clear cell carcinoma, endometrioid carcinoma, endometriosis and benign endometrium were subjected to an in-depth liquid chromatography-tandem mass spectrometry analysis on the Q-Exactive Plus. Protein identification and quantification were performed using MaxQuant, while downstream analyses were performed using Perseus and various bioinformatics databases. Results: Approximately 9000 proteins were identified in total, representing the first in-depth proteomic investigation of endometriosis and its associated cancers. This proteomic data was shown to be biologically sound, with minimal variation within patient cohorts and recapitulation of known markers. While moderate concordance with genomic data was observed, it was shown that such data are limited in their abilities to represent tumours on the protein level and to distinguish tumours from their benign precursors. Conclusions: The proteomic data suggests that distinct markers may differentiate endometrioid and clear cell carcinoma from endometriosis. These markers may be indicators of pathobiology but will need to be further investigated. Ultimately, this dataset may serve as a basis to unravel the underlying biology of the endometrioid and clear cell cancers with respect to their endometriotic origins